Antibacterial Properties of Biobased Polyester Composites Achieved

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2262−2270

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Antibacterial Properties of Biobased Polyester Composites Achieved through Modification with a Thermally Treated Waste Scallop Shell Chin-San Wu,*,† Dung-Yi Wu,‡ and Shan-Shue Wang† †

Department of Applied Cosmetology, Kao Yuan University, Kaohsiung County, Taiwan 82101, Republic of China Department of Materials Science and Engineering, Cornell University, Ithaca, New York 14850, United States

‡

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S Supporting Information *

ABSTRACT: Novel antibacterial properties of composites prepared from thermally treated waste white scallop shell powder (TWWSSP) and modified polylactide (MPLA) are reported. The waste shell (calcium carbonate, CaCO3) was calcined at 1000 °C to completely form calcium oxide (CaO) and calcium hydroxide (Ca(OH)2). The composition and structure of the calcined product were characterized using energy dispersive spectrometry, Fourier transform infrared spectroscopy, and X-ray diffraction. The TWWSSP was studied to determine its effectiveness as a bactericidal agent when incorporated into MPLA to form composites. Infrared, tensile, and morphological characterizations indicated an enhanced adhesion between the TWWSSP and the MPLA in the composites and an improved compatibility compared with the PLA/WWSSP composites. The MTT assay and cell adhesion tests on the composites revealed that the relative growth rate of Mus dunni fibroblast (MDFB) cells increased with an increasing TWWSSP content, which indicated that the composites were not cytotoxic. Moreover, TWWSSP containing CaO and Ca(OH)2 enhanced the antibacterial activity of the composites; MPLA composites that contained TWWSSP had a better antibacterial activity. The antibacterial and biodegradable properties of the MPLA/TWWSSP and PLA/WWSSP composites have a great potential for many applications, especially food packaging and biomedical materials. KEYWORDS: antibacterial activity, biocompatibility, biodegradation, polymer−matrix composites, waste white scallop shell 800 °C; the residue was then mixed with plastics to make bacteriostatic food packing materials, thereby prolonging the food freshness time. The combined development of environmentally friendly biobased polyester and shellfish waste is a topic of great interest from the perspectives of ecology and the recycling of renewable resources.15−17 Among numerous biobased polyesters, polylactide (PLA)-based ones are the most mature; they exhibit a good processability, mechanical performance, biocompatibility, and biodegradability.18,19 Currently, PLA is the biobased polyester most extensively used for biomedical packaging materials, thin-film materials, and wound dressings.20,21 However, PLA is not antibacterial. To provide this property, PLA has been mixed with antimicrobial agents to form antibacterial composites. Scaffaro et al.22 doped graphene nanoplatelets and ciprofloxacin into PLA, obtaining an antibiotic activity against Micrococcus luteus. Han et al.23 prepared a composite film of PLA and chitosan, which greatly diminished the activity of Escherichia coli (E. coli), making it of interest for biomedicine and food packaging applications.

1. INTRODUCTION Many countries are advancing environmental protection and resource recycling policies.1,2 Abundant marine shellfish substances (e.g., clam shells, oyster shells, and scallop shells)3−5 are generated in large quantities as byproducts of the marine product processing industry.6,7 However, shellfish waste contains substantial amounts of calcium carbonate (CaCO3) and residual meat, and the latter is likely to be decomposed by microorganisms into substances harmful to human health (e.g., pathogens causing diarrhea, malaria, and dengue fever).8,9 Furthermore, the decomposition exudes harmful gases, such as hydrogen sulfide and ammonia, leading to environmental issues.10,11 To solve these issues, calcination has been used to eliminate unwanted organic matter from shellfish waste; the main constituent CaCO3 is thermally treated to form calcium hydroxide (Ca(OH)2) or calcium oxide (CaO), which can be recycled or used in the production of high-value materials (e.g., biodiesel catalysts, antibiotics, and adsorbents). Kouzuet et al.12 calcined discarded scallop shells at 900 °C to produce a biodiesel catalyst for the preparation of colza oil. Khan et al.13 calcined abandoned oyster shells at 900 °C and then subjected the product to an acid−alkali treatment; the calcined powder has potential applications in coating and antibiotic materials. Kao et al.14 calcined spent clam shells at © 2019 American Chemical Society

Received: March 13, 2019 Accepted: April 25, 2019 Published: April 26, 2019 2262

DOI: 10.1021/acsabm.9b00205 ACS Appl. Bio Mater. 2019, 2, 2262−2270

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ACS Applied Bio Materials

Scheme 1. Schematic Diagram of Polylactide Modified with Thermally Treated Waste White Scallop Shell Powder and the Fabrication of Composites

age)25 was applied as an MPLA compatibilizer. Table S1 provides the compositions of the composites prepared in this study. The MPLA/TWWSSP composites were extruded at 180−185 °C and 50 rpm to prepare the three-dimensional (3D)-printed filaments. The diameter of these filaments was maintained at 1.75 ± 0.05 mm by adjusting the coiler speed. The fabrication of the 3D-printed filaments and their products is shown in Scheme S1.26 2.3. Characterization. The wide-angle X-ray diffractometer (XRD; model D/max 2500; Rigaku, Tokyo, Japan) used a Cu target and Kα radiation (λ = 1.5406 Å) and operated at a scanning rate of 2° min−1 under 40 kV and 20 mA. Data were collected in the 2θ range 5°−70°. Samples were also analyzed from 400 to 4000 cm−1 at a resolution of 2 cm−1 using a Fourier transform infrared spectrometer (FT-IR; Bio-Rad, Hercules, CA, USA). Samples were mixed with KBr, and the resultant mixture was pressed into films. The morphology of TWWSSP and its composites was evaluated using scanning electron microscopy (SEM; model S3000N; Hitachi, Tokyo, Japan).25 The tensile properties of samples were measured using a universal testing machine.26 The surface elements of gold-coated WWSSP and TWWSSP were identified by energy dispersive spectrometry.27 Thermogravimetric analysis was performed with a sample weight of 5−8 mg using a thermogravimetric analyzer.28 Samples were heated at 10 °C min−1 from 30 to 700 °C under a nitrogen atmosphere, and the initial decomposition temperature (IDT) of the sample was then determined. The wettability of PLA/WWSSP and MPLA/TWWSSP composite surfaces was determined by measuring the contact angle using the sessile drop method with a goniometer.26 All measurements were performed at room temperature (controlled at 30 °C) using distilled water as follows. Water was injected at 0.6 μL s−1, and the drop image was taken 1 min after detection of the contact angle. The contact angles were measured for at least five points; the average is reported. Single-layer printing (ca. 0.05 mm thick) was performed using a material extrusion 3D printer with x-axis and z-axis moving velocities of 150 and 23 mm s−1, respectively.26 2.4. In Vitro Cytotoxicity Testing. Mus dunni normal tail fibroblasts (MDFB) were grown in a culture, as reported previously.29 The proliferation of MDFB cells on a membrane was evaluated. Each well of a 24-well plate was embedded with 3 × 104 cells and incubated for 1, 3, and 5 days. The cell survival was quantitatively counted by MTT analysis.29 The morphology of the various sample films was studied by SEM to better understand the growth condition and morphology of the MDFB cells. A sample was sterilized and loaded into 24 wells. Each well was then filled with 9 × 103 cells and incubated for 60 h. The cells were then fixed with 5% glutaraldehyde solvent, and the film surface was washed with PBS and ultrapure water to remove salts. After the cells were completely fixed, the samples

Kostic et al.24 fabricated alginate microbeads containing colloidal silver (Ag) nanoparticles in a PLA matrix. Microbead films of the PLA/Ag/alginate multifunctional ternary composite prevented the growth of Staphylococcus aureus (S. aureus). Herein, we report combining a thermally treated waste white scallop shell (WWSS) with PLA or modified PLA (MPLA) to form composites. Calcining the WWSS at 1000 °C transformed the CaCO3 constituent into Ca(OH)2 and CaO. Combining this with MPLA provided the composite with excellent antibacterial and biocompatibility properties. The adhesion, tensile strength, and thermal properties of the composite were improved by modifying the interface between MPLA and TWWSS with a compatibilizing agent. Such antibacterial composites have potential applications in antibacterial films, packaging materials, and foam sheets.

2. EXPERIMENTAL METHODS 2.1. Materials. Polylactide (PURAPOL L130; density 1.24 g/cm3; melt flow index 2.16 kg at 190 °C when measured according to the standard ISO 1133-A) was supplied by Corbion Co., Ltd. (Amsterdam, The Netherlands). Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolelium bromide (MTT) were obtained from Sigma-Aldrich Chemicals Inc. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), nutrient broth, and agar were obtained from Gibco-BRL (Gaithersburg, MD, USA). All buffers and other reagents were of the highest purity grade commercially available. 2.2. Fabrication of the Composites and 3D-Printed Filaments. The WWSS was obtained from Xingda Fishing Port in Kaohsiung (Taiwan). The collected WWSS was brushed with water to eliminate surface dirt and impurities. It was then laid to dry in a vacuum oven at 90−100 °C for 1 day to reduce the water content to less than 0.1%. The dried shells were then ground, and the output was sieved through 800−2500 mesh screens to collect a 5−15 μm fraction of waste white scallop shell powder (WWSSP). The fraction was then dried for 12 h at 100−110 °C in a vacuum oven. A high-temperature calcinator was used for further thermal treatment of the WWSSP; the temperature was controlled to 250, 500, 750, and 1000 °C for 2 h of calcination. The corresponding thermally treated waste white scallop shell powders (TWWSSPs) were produced when the calcined WWSSP was cooled to an ambient temperature. The overall TWWSSP processing procedure is illustrated in Scheme 1. PLAgrafted maleic anhydride (PLA-g-MA; 0.98 wt % grafting percent2263


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ACS Applied Bio Materials were soaked in different alcohol concentrations (15, 25, 35, 75, 85, and 95%) for about 5 min. The alcohol was then completely removed by evaporation. The adhesion of the MDFB cells in the various sample films was observed. The effect of materials on the cell cycle was determined by flow cytometry with propidium iodide (PI) dye. The composites were placed in 6 cm diameter culture dishes and then were UV-sterilized for 1 h. Initially, 2 × 105 MDFB cells were cultivated with the composites in the culture dishes for 24 h. Then, the cells were harvested and fixed in 70% ice alcohol for 8 h to disrupt the cell membrane. After the supernatant was removed by centrifugation, the cells were exposed to a PI/Triton X-100 DNA staining solution for 0.5 h in a dark environment at room temperature. The cells were then analyzed by flow cytometry and FL2 PMT. The cell distribution in the cell cycle was examined with WinMDI software (version 2.8).29 2.5. Antibacterial Assay. The antibacterial activity of WWSSP, TWWSSP, and the composites was examined against E. coli (Gramnegative bacteria) and S. aureus (Gram-positive bacteria) (Bioresource Collection and Research Center). Both E. coli and S. aureus bacteria were activated and grown in liquid media for 24 h. Bacterial solution (100 μL) was dropped onto the culture dish and then spread across the dish.26 The materials used in the antibacterial test were prepared as a tablet having a diameter of 1.15 cm and a thickness of 0.05 cm by pressing 0.1 g of WWSSP or TWWSSP powder with a tablet press at 5000 psi. Film samples (0.05 cm thick) of PLA/ WWSSP and MPLA/TWWSSP were produced using a hot press and then cut into circular films of a diameter of 1.15 cm. Then, a sample was washed with 75% alcohol, exposed to ultraviolet light, and placed at the center of the culture dish. The culture dish was sealed with elastic adhesive tape and kept in the incubator at 37 °C for 18 h. The antibacterial activity was examined by the inhibition zone.26 For the antibacterial quantification test, the E. coli bacterial liquid (25 mL; 2 × 105 CFU mL−1) was cultivated with a 1 g sample in a flask at 37 °C for 24 h in an incubator. Then, the sample solution (100 μL) was dropped into a culture dish, spread, and allowed to propagate for 18 h. The bacteria were then counted. 2.6. Biodegradation Test. The biodegradability was evaluated by examining the weight loss of the various composites over time in soil. Sample membranes (35 × 25 × 0.5 mm3) were prepared, buried underneath 10−12 cm of alluvial soil in boxes, and maintained at 35− 50% relative humidity and 25−35 °C for 120 days. The samples were recovered every 10 days, washed, and equilibrated in a desiccator. Then, the samples were weighed and reburied.30 2.7. Statistical Analysis. Data were analyzed using Sigma Plot 10.0 software (Systat Software Inc., San Jose, CA, USA) with a twotailed Student’s t test. The results represent at least five replicate experiments and are reported as the mean ± standard deviation (SD). A p value less than 0.05 was considered significant for all tests.

Figure 1. Wide-angle X-ray diffraction patterns of (a) untreated waste white scallop shell powder (WWSSP) and treated waste white scallop shell powders (TWWSSPs) formed at (b) 500 and (c) 1000 °C. Signals: 1:17.9°, 2:34.2°, 3:28.6°, 4:32.1°, 5:34.5°, 6:37.2°, 7:47.3°, 8:50.7°, and 9:53.8°.

WWSSP from CaCO3 to CaO and Ca(OH)233,34 with simultaneous CO2 release. We chose TWWSSP formed at 1000 °C as the filler for our composites because CaO and Ca(OH)2 are established antibacterial compounds. Figure 2 illustrates the FT-IR spectra of the pristine WWSSP and that thermally treated at 500 and 1000 °C to form the

3. RESULTS AND DISCUSSION 3.1. Characterization of White Scallop Shell Powder. Figure 1 shows the crystalline XRD patterns of the original WWSSP and TWWSSPs obtained after thermally treating the WWSSP at 500 and 1000 °C. The XRD peaks of the original WWSSP (Figure 1a) exhibited eight peaks at 23.1°, 29.4°, 31.2°, 35.9°, 39.1°, 43.1°, 47.8°, and 48.6°, which indicated that it was primarily CaCO3.31 Comparing the XRD patterns of the original WWSSP and the TWWSSP prepared at 500 °C (Figure 1b) indicates that there are two new peaks at about 17.9° (1) and 34.2° (2), respectively. This result is consistent with partial decomposition of CaCO3 into Ca(OH)2 during the thermal treatment of WWSSP at 500 °C.32 Compared with Figure 1c, seven new peaks are apparent at 28.6° (3), 32.1° (4), 34.5° (5), 37.2° (6), 47.3° (7), 50.7° (8), and 53.8° (9) in the XRD pattern of the TWWSSP formed at 1000 °C (Figure 1c). These results indicate that increasing the calcination temperature to 1000 °C led to complete transformation of

Figure 2. Fourier transform infrared spectra of (a) untreated WWSSP and TWWSSP formed at (b) 500 and (c) 1000 °C.

corresponding TWWSSPs. Characteristic peaks of the pristine WWSSP appear at 716 (CO3, symmetric), 872 (CO3, asymmetric), 1486 (CO3, asymmetric), 2512 (CO3, asymmetric), and 2920 and 3318 cm−1 (OH) (Figure 2a). Similar data have been published elsewhere.35,36 The peaks of TWWSSP formed at 500 °C (Figure 2b) are significantly different in position and intensity from those of WWSSP. Notably, the peaks observed at 1486 and 3318 cm−1 for WWSSP are shifted to 1436 and 3341 cm−1. These bands are assigned to the stretching vibrations of the OH groups of Ca(OH)2 and the H−O−H bending modes of the hydroxyl group,37 respectively, and are consistent with Ca(OH)2 formation and H2O elimination. Figure 2c shows the FT-IR 2264


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The OH stretching vibration peak at 3200−3700 cm−1 became more intense in the PLA/WWSSP (3 wt %) composite (Figure 3c), and an additional shoulder appeared at 3332 cm−1 due to the OH groups of WWSSP. For the PLA/WWSSP (3 wt %) and MPLA/TWWSSP (3 wt %) composites (Figure 3c,d), the vibration band broadened and moved from 3332 to 3368 cm−1. The above observations indicate the presence of strong hydrogen bond formation between the anhydride carboxylic groups of the MPLA and the OH groups of the TWWSSP. Figure 3d shows two peaks at 582 and 1735 cm−1, not found in the PLA/WWSSP (3 wt %) spectrum, which were observed in the MPLA/TWWSSP (3 wt %) FT-IR spectrum. These peaks were assigned to CaO bond stretching modes42 and the ester stretching vibration27 of MPLA/TWWSSP (3 wt %), respectively. 3.3. Morphology, Tensile Properties, and Thermal Properties of White Scallop Shell Powder and the Composites. Figure 4a,b shows SEM images of fractured surfaces of PLA/WWSSP (3 wt %) and MPLA/TWWSSP (3 wt %) specimens. Figure 4a reveals that the WWSSP was poorly adhered throughout the PLA matrix in PLA/WWSSP (3 wt %), with many WWSSP particles incompletely sheathed by the PLA. Externally exposed WWSSP indicated a poor wettability between WWSSP and PLA, which limited interfacial adhesion. The enhanced interfacial adhesion in MPLA/TWWSSP (3 wt %) was attributed to the improved compatibility between the TWWSSP and the MPLA matrix because of chemical bond formation between them (Figure 4b). The tensile strength (TS) at the break and Young’s modulus of PLA/WWSSP and MPLA/TWWSSP specimens is presented in Figure 4c,d, respectively, as a function of WWSSP and TWWSSP content. The TS at the break and Young’s modulus of original PLA (40.8/3550 MPa) decreased slightly after the PLA was modified to form MPLA (40.5/3500 MPa) and that of the PLA/WWSSP specimens decreased continuously with an increasing WWSSP content (from 40.8/ 3550 to 32.1/2820 MPa). Poor adhesion of WWSSP to the PLA matrix was responsible for this behavior and had an obvious effect on the tensile properties of the composites. The TS at the break and Young’s modulus of the MPLA/TWWSSP composites (Figure 4c,d) increased with an increasing TWWSSP content. However, for the TWWSSP content beyond 3 wt %, these properties decreased with an increasing TWWSSP content, which was attributed to aggregation of the TWWSSP particles. The thermal stability of WWSSP and TWWSSP plays an important role in the processing of PLA and modified MPLA, respectively. Thermogravimetric analysis of the PLA/WWSSP and MPLA/TWWSSP composites provided details of their thermal stability. Figure 5 and Table 1 indicate the effect of WWSSP and TWWSSP on the IDT at 5% weight loss (defined herein as the IDT5%). After WWSSP and TWWSSP were loaded into the PLA and MPLA matrix, respectively, the thermal stability clearly increased. The decomposition temperature of the composites shifted to higher values with an increasing filler addition, confirming the roles of WWSSP and TWWSSP in strengthening the PLA and MPLA composites, respectively.43,44 With the same WWSSP and TWWSSP content, the IDT5% of PLA/WWSSP was lower than that of the MPLA/TWWSSP composite. The lower IDT5% of the PLA/WWSSP composite was due to an increased inhibition of the polymer chain movement by WWSSP and/or a bonding

spectrum of the TWWSSP formed at 1000 °C. There are three peaks at 578, 1468, and 3648 cm−1 that are not present in the spectra of WWSSP and TWWSSP formed at 500 °C. These peaks were assigned to CaO, as reported elsewhere.38 The results are consistent with the formation of CaO during the formation of TWWSSP at 1000 °C. Figure S1 shows SEM micrographs and EDS analyses of the original WWSSP and TWWSSPs produced at 500 and 1000 °C. The untreated WWSSP surface was covered with impurities (Figure S1a), while the TWWSSPs formed at 500 and 1000 °C exhibited a cleaner and smoother appearance (Figure S1b,c). Additionally, the surface is smoother in Figure S1b than in Figure S1c. Figure S1d−f shows the results of EDS composition analyses of WWSSP and TWWSSPs prepared at 500 and 1000 °C. Figure S1d shows that the WWSSP contained C, O, and Ca. The TWWSSP formed at 500 °C also contained these elements, but the C and Ca components were 6.2% lower and 6.5% higher, respectively, than that of WWSSP. The TWWSSP formed at 1000 °C only contained O and Ca because the main component of the original WWSSP was CaCO3. The main CaCO3 component of the WWSSP was partially pyrolyzed at 500 °C and converted into Ca(OH)2. During the treatment at 1000 °C, the main CaCO3 component of the WWSSP was completely pyrolyzed to Ca(OH)2 and CaO,39,40 in agreement with the aforementioned structural identification by FT-IR and XRD. 3.2. Structure of White Scallop Shell Powder and Its Composites. The absorption bands at 3200−3700, 1660− 1770, and 500−1500 cm−1 corresponding to PLA were observed in the spectra of both polymers (Figure 3a,b). Two extra shoulders at 1783 and 1851 cm −1 corresponding to MPLA are characteristic of maleic anhydride groups (Figure 3b). Similar results have been described elsewhere.41

Figure 3. Fourier transform infrared spectra of (a) polylactide (PLA), (b) modified polylactide (MPLA), (c) PLA/WWSSP (3 wt %), and (d) MPLA/TWWSSP (3 wt %). 2265


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Figure 4. Scanning electron microscopy images showing the distribution and wetting of (a) PLA/WWSSP (3 wt %) and (b) MPLA/TWWSSP (3 wt %) (using TWWSSP formed at 1000 °C). Tensile properties of PLA/WWSSP and MPLA/TWWSSP: (c) tensile strength at the break and (d) Young’s modulus.

Table 1. Effect of WWSSP or TWWSSP Content on the Initial Decomposition Temperature at 5% Weight Loss (Initial Decomposition Temperature, IDT5%) and Char Residual Yield of PLA/WWSSP and MPLA/TWWSSP Composites PLA/WWSSP

Figure 5. Thermogravimetric curves for PLA, PLA/WWSSP, and MPLA/TWWSSP composites.

MPLA/TWWSSP

WWSSP or TWWSSP (wt %)

IDT5% (°C)

weight retention (%) at 500 °C

IDT5% (°C)

weight retention (%) at 500 °C

0 1 2 3 4 5

295 303 318 328 332 335

0.3 1.8 3.7 6.1 7.3 8.3

292 316 343 366 372 378

0.2 3.2 7.1 10.3 11.9 13.7

composite. This result was attributed to TWWSSP preventing the transport of thermal decomposition in the polymer composite.47 The TGA results show that incorporation of a small amount of TWWSSP can significantly enhance the thermal stability of MPLA/TWWSSP composites. 3.4. Surface Hydrophilicity. Figure 6 illustrates how water drop contact angle measurements were used to assess the hydrophilic characteristics of PLA, MPLA, and the composites. The contact angles of the original PLA and MPLA surfaces were 83.95° and 82.54°, respectively. With 3 wt % WWSSP and 5 wt % TWWSSP, the contact angles of the PLA/WWSSP and MPLA/TWWSSP composites were reduced from 78.13° to 74.45° and 73.87° to 66.11°, respectively. These reductions were attributed to microgaps/defects in the matrix between polymer chains and WWSSP or TWWSSP,48,49 as well as to the presence of hydrophilic substances.

effect, which resulted in an increased interfacial adhesion of TWWSSP in MPLA compared with WWSSP in PLA.45,46 The IDT5% values for the MPLA/TWWSSP composites were about 13−43 °C higher than those of the PLA/WWSSP composites; the increment in IDT5% was 74 °C for 3 wt % TWWSSP but only 12 °C for 5 wt % TWWSSP. The optimal loading of TWWSSP confirmed in this study was 3 wt %; excess TWWSSP was detrimental to the thermal stability due to the separation of organic and inorganic phases. Figure 5 and Table 1 also indicate that the char residual yield of the MPLA/TWWSSP composite increased with an increasing TWWSSP content, consistent with retardation of the thermal decomposition of MPLA in the MPLA/TWWSSP 2266


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Figure 6. Contact angle profiles for PLA, MPLA, PLA/WWSSP, and MPLA/TWWSSP composites.

Furthermore, the contact angles of teh PLA/WWSSP and MPLA/TWWSSP composites with the same WWSSP and TWWSSP content were compared; the contact angle of MPLA/TWWSSP was lower by 4.26° at 3 wt % and 8.34° at 5 wt % than the values of PLA/WWSSP. Additionally, the MPLA/TWWSSP composites had lower contact angles than the PLA/WWSSP composites with the same WWSSP and TWWSSP content. This contact angle reduction was ascribed to the surface hydrophilicity of CaO and Ca(OH)2 present in the TWWSSP, which facilitated water molecule penetration. Therefore, MPLA/TWWSSP displayed a better surface hydrophilicity. 3.5. Cell Proliferation, Cell Dispersion, and Cell-Cycle Progression Assessment. The MDFB cell growth of all composites increased with an increasing incubation time, indicating a good biocompatibility and no cytotoxicity of the composites (Figure 7a). Additionally, the cell viability of the PLA/WWSSP and MPLA/TWWSSP composites increased considerably with an increasing amount of WWSSP and TWWSSP, respectively. Large loadings of WWSSP and TWWSSP in PLA or MPLA, respectively, provided an excellent biological activity.50,51 Figure 7b illustrates the cell adhesion morphology after 60 h of culture of MDFB cells on a film sample. The SEM images show that the cells had good growth in the control group, PLA, PLA/WWSSP (3 wt %), and MPLA/TWWSSP (3 wt %). MPLA/TWWSSP (3 wt %) displayed relatively complete cell adhesion, while PLA and PLA/WWSSP (3 wt %) had slightly less cell aggregation than the control group. Good cell proliferation was achieved by adding 3 wt % TWWSSP, with a good hydrophilicity and bioactive molecules, to the MPLA/ TWWSSP complex.52 Figure 7 shows that the PLA/WWSSP and MPLA/TWWSSP composites provided an excellent environmental matrix for MDFB cell growth. The MDFB cell cycles affected by various samples are shown in Figure S2. There was no significant difference (p < 0.05) between the growth period (G1), synthesis phase (S), or mitosis period (G2/M) phase and the control in terms of cell numbers (events) versus DNA content (FL2-A) (Figure S2a) or the total cell population (Figure S2b). Therefore, the various composite materials did not significantly affect the MDFB cell cycle; moreover, the PLA/WWSSP and MPLA/

Figure 7. (a) Cell viability of Mus dunni mouse normal tail fibroblasts (MDFB) seeded on no PLA (control), PLA, PLA/WWSSP (1, 3, 5 wt %), and MPLA/TWWSSP (1, 3, 5 wt %) by an MTT assay. (b) Scanning electron microscope images of MDFB cells showing their dispersion and adhesion on no PLA (control), PLA, PLA/WWSSP (3 wt %), and MPLA/TWWSSP (3 wt %) composites after 72 h of culture. Statistical analyses were performed using t tests, with significant differences (*) determined at the p < 0.05 level for each treatment versus the control group.

TWWSSP composites did not damage cells and exhibited a good biocompatibility.53 3.6. Antibacterial Activity and Biodegradation Behavior. Figure 8 shows the zones of inhibition against E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) in culture plates containing untreated (1) and thermally treated WWSSP at 250 (2), 500 (3), 750 (4), and 1000 °C (5). Both untreated WWSSP and TWWSSP (250 °C) displayed no zones of inhibition. However, there were 0.19/ 0.14, 0.39/0.31, and 0.81/0.73 cm bacterial inhibition zones in the experiments of E. coli/S. aureus cells cocultured with TWWSSP formed at 500, 750, and 1000 °C, respectively. Thermal treatment above 500 °C yielded a TWWSSP with antibacterial activity. Additionally, the TWWSSP formed by treatment of WWSSP at 1000 °C yielded the largest inhibition zone of the various TWWSSPs. This phenomenon is attributed to the composition of TWWSSP, which is only Ca(OH)2 and CaO after thermal treatment at 500 to 1000 °C. We chose 1000 °C as the treatment temperature to prepare the composites reported herein. The zones of inhibition for PLA (6), PLA/WWSSP (7, 8), and MPLA/TWWSSP (9, 10) composites in a culture plate containing E. coli and S. aureus are shown in Figure 8. The figure shows that PLA (6), and the PLA/WWSSP (3 wt %) (7) 2267


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5 wt % MPLA/TWWSSP. The degradation rate of PLA/ WWSSP was thus greater than that of MPLA/TWWSSP. Chemical bonds formed between MPLA and WWSSP compacted the material structure, thereby restricting the approach of water molecules and microorganisms and slowing down the degradation rate in the soil environment.

4. CONCLUSIONS Structural analysis using XRD, FT-IR, and EDS, and antibacterial activity testing indicated that TWWSSP exhibited antibacterial properties, particularly those TWWSSPs that had been formed by calcining WSSP at 1000 °C, as well as blends of TWWSSP with MPLA. The fabrication, structural, antibacterial, and biological properties of composites made from MPLA/TWWSSP were investigated. Infrared spectra were consistent with chemical reactions having occurred between TWWSSP and maleic anhydride groups during MPLA formation. There was good adhesion between the TWWSSP and MPLA matrix in the MPLA/TWWSSP composites. The improved adhesion enhanced the TS at the break and the Young’s modulus. Moreover, increasing the TWWSSP content in the MPLA/TWWSSP composites improved cell growth. A strong inhibitory effect against E. coli and S. aureus bacteria was observed with the MPLA/ TWWSSP composites. Although soil tests showed an increase in WWSSP or TWWSSP content, the degradation rate of the composites increased. The MPLA/TWWSSP composite degraded more slowly than PLA/WWSSP but was still significantly faster than that for pure PLA or MPLA. Ideally, a new commercial material must have adequate tensile and biodegradable properties. The MPLA/TWWSSP composites had good elongation and biodegradable and antibacterial properties and are straightforward to manufacture. These properties make MPLA/TWWSSP composites promising for industrial applications in tissue engineering, antibacterial materials or products, and air filtration membranes.

Figure 8. Zones of inhibition for WWSSP, TWWSSP, PLA/WWSSP, and MPLA/TWWSSP composites in bacterial culture plates (Escherichia coli and Staphylococcus aureus). WWSSP without treatment (1) and with treatment to form TWWSSP at 250 (2), 500 (3), 750 (4), and 1000 °C (5). PLA (6), PLA/WWSSP 3 wt % (7), PLA/WWSSP 5 wt % (8), MPLA/TWWSSP 3 wt % (9), and MPLA/TWWSSP 5 wt % (10).

■

and PLA/WWSSP (5 wt %) (8) composites, had no bacterial inhibition zone for E. coli and S. aureus. This revealed that the PLA and PLA/WWSSP composites did not have an antibacterial effect. However, there were 0.29/0.21 and 0.45/ 0.34 cm inhibition zones in E. coli/S. aureus cells of the MPLA/ TWWSSP (3 wt %) (9) and MPLA/TWWSSP (5 wt %) (10) composites, respectively. These results likely resulted from the presence of Ca(OH)254,55 and CaO,56,57 and more specifically from the release of hydroxyl ions and active oxygen species generated at the TWWSSP surface, which inactivated the microorganisms. Figure S3 shows the quantitative antibacterial activity for PLA, MPLA, and their composites. Neither PLA, MPLA, nor PLA/WWSSP had antibacterial properties, which is attributed to the PLA and WWSSP composition. However, the antibacterial activity of the MPLA/TWWSSP composites increased remarkably with an increasing TWWSSP content. Notably, all bacteria were killed when the content was above 3 wt %. TWWSSP is a superior antibacterial additive for MPLA. Figure S4 shows the correlation between the weight loss of PLA, MPLA, and its composites and the burial time in soil. During the first 60 days, all tested substances degraded at a rapid rate; after 60 days, the weight loss rate decreased. Composites containing WWSSP or TWWSSP clearly lost weight. When the WWSSP or TWWSSP content is greater, the weight loss is more rapid. During the first 60 days, the weight loss of 5 wt % PLA/WWSSP was most noticeable, followed by

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00205. 3D-printed filaments and their products, as well as their surface morphology (as revealed by SEM imaging), and quantitative EDS analyses of WWSSP and TWWSSP formed at 500 and 1000 °C; formulation preparation and the quantitative antibacterial activity and biodegradative behavior of the PLA/WWSSP and MPLA/ TWWSSP composites (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Tel: +886-7-6077685. Fax: +886-7-6077788. ORCID

Chin-San Wu: 0000-0002-1498-8588 Dung-Yi Wu: 0000-0002-3749-9429 Shan-Shue Wang: 0000-0001-6187-034X Notes

The authors declare no competing financial interest. 2268


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ACKNOWLEDGMENTS The author thanks the Ministry of Science and Technology (Taipei City, Taiwan, R.O.C.) for financial support (MOST106-2622-E-244-002-CC3).

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