Graphical abstract
Abstract
Objectives
This study reports on the synthesis, materials characterization, antimicrobial capacity, and cytocompatibility of novel ZnO-loaded membranes for guided tissue/bone regeneration (GTR/GBR).
Methods
Poly(ɛ-caprolactone) (PCL) and PCL/gelatin (PCL/GEL) were dissolved in hexafluoropropanol and loaded with ZnO at distinct concentrations: 0 (control), 5, 15, and 30 wt.%. Electrospinning was performed using optimized parameters and the fibers were characterized via scanning and transmission electron microscopies (SEM/TEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), contact angle (CA), mechanical testing, antimicrobial activity against periodontopathogens, and cytotoxicity test using human dental pulp stem cells (hDPSCs). Data were analyzed using ANOVA and Tukey ( α = 5%).
Results
ZnO nanoparticles were successfully incorporated into the overall submicron fibers, which showed fairly good morphology and microstructure. Upon ZnO nanoparticles’ incorporation, the PCL and PCL/GEL fibers became thicker and thinner, respectively. All GEL-containing membranes showed lower CA than the PCL-based membranes, which were highly hydrophobic. Overall, the mechanical properties of the membranes were reduced upon ZnO incorporation, except for PCL-based membranes containing ZnO at the 30 wt.% concentration. The presence of GEL enhanced the stretching ability of membranes under wet conditions. All ZnO-containing membranes displayed antibacterial activity against the bacteria tested, which was generally more pronounced with increased ZnO content. All membranes synthesized in this study demonstrated satisfactory cytocompatibility, although the presence of 30 wt.% ZnO led to decreased viability.
Significance
Collectively, this study suggests that PCL- and PCL/GEL-based membranes containing a low content of ZnO nanoparticles can potentially function as a biologically safe antimicrobial GTR/GBR membrane.
1
Introduction
Periodontitis is a very prevalent (ca. ∼50% of the adult population) and aggressive dental pathology affecting both the gingiva and anchoring tissues of the tooth . It is a bacterial-mediated inflammatory process, and if not treated, it may progressively destroy periodontal tissues, eventually resulting in tooth loss . A recent systematic review confirmed that severe periodontitis has increased in the past two decades, and a predictable increasing burden of the disease can be expected, since life expectancy among the world population is continuously growing .
The traditional clinical treatment of periodontitis consists of surgical procedures and mechanical debridement (i.e. scaling and root planing), which allows healing of periodontal pockets/defects . Moreover, systemic and/or local antibiotic therapy may also be indicated in order to combat the infection or re-colonization of periodontopathogens . Nonetheless, depending on the size of the pocket/inflamed site, the complete regeneration of periodontal tissues remains a difficult clinical task . In this way, regenerative approaches, such as the use of bioresorbable or non-resorbable barrier membranes employed in so-called guided tissue/bone regeneration (GTR/GBR) strategies, as well as emerging technologies (e.g. cell-based and gene therapies, the use of bone anabolics, and laser treatments, among others) have been investigated in terms of their clinical potential in periodontal tissue regeneration . Among the aforementioned approaches, GTR/GBR membranes present the unique characteristic of resembling the extracellular matrix (ECM), thus facilitating cell adhesion, proliferation, and the differentiation of new tissues . Furthermore, GTR/GBR membranes can be synthesized using a variety of natural and/or synthetic polymers, and depending on processing conditions, bioactive (e.g. antimicrobials) fibers on the nano- and micro-scale range can be fabricated .
Despite the fairly significant clinical success achieved with the use of membranes in GTR/GBR strategies, tissue regeneration is strongly dependent on the absence of infection, and therefore, the proper maintenance of a bacteria-free environment . To that end, several controlled-release systems, such as Atridox ® (doxycycline-based) and PerioChip ® (chlorhexidine-based), have been thoroughly used to locally deliver antimicrobial agents to the periodontal pocket, resulting in positive clinical effects . More recently, antimicrobial agents have been incorporated into GTR/GBR membranes, endowing to them antibacterial activity . Notwithstanding, it is well recognized that antibiotics may produce some important side-effects, mainly those related to bacterial strain resistance, which is a current global concern, since bacteria are becoming resistant to several antibiotic therapies . Therefore, the development of biomaterials for GTR/GBR applications based on alternative antibacterial agents rather than antibiotics is paramount to translate safer regenerative technologies into clinical practice.
Inorganic ions and metallic oxides have extensively demonstrated antibacterial properties . Silver is the most effective antibacterial metal at low concentrations and against both gram-positive (G+) and gram-negative (G−) bacteria. Meanwhile, several other metal oxides (e.g. copper, magnesium, calcium, titanium, and zinc) have also displayed important antibacterial activity against a wide variety of microorganisms. Zinc oxide (ZnO), for example, is one of the few zinc-based compounds listed as being generally recognized as a safe (GRAS) material by the Food and Drug Administration (FDA) . Even though the mechanism of action is still vaguely understood, ZnO has been successfully used as an antibacterial agent in food packaging , restorative dental materials , wound dressings , and in tissue engineering applications . Indeed, electrospun polymer-based scaffolds/membranes loaded with ZnO have recently displayed not only antibacterial properties , but also enhanced cell proliferation/wound healing . It is also important to note that the antibacterial activity of these ZnO-loaded membranes was found to be dose-dependent (≥5 wt.%) . Nonetheless, the earlier study tested the ZnO-scaffolds only on Staphylococcus aureus (G+) and Escherichia coli (G−), thus further highlighting the need to investigate their potential against periodontopathogens based on their application as GTR/GBR membranes.
It is well-established that degradability is one of the most important aspects involved in the clinical success of bioresorbable GTR/GBR membranes . Indeed, it is expected that the membrane would be completely resorbed after treatment is completed, thus eliminating an additional surgical procedure for its removal from the regenerated site . The degradability rate of GTR/GBR membranes strongly depends on the type of polymer system used. Poly(ɛ-caprolactone) (PCL) is a synthetic polymer with known degradability and hydrophobic characteristics , and considering the latter property influences the former, with highly hydrophobic systems displaying lower degradability, PCL usually presents a moderate degradation rate. Recent research has utilized the strategy of blending natural polymers, such as collagen or gelatin, with PCL to grant enhanced degradability and hydrophilicity , as well as better cell-membrane response .
In this study, we tested the influence of ZnO nanoparticles and gelatin on the physico-chemical and mechanical properties of PCL-based electrospun membranes. Furthermore, we report for the first time on the antibacterial activity and cytocompatibility of these membranes’ targeting a specific application in periodontal tissue regeneration by careful antimicrobial and cell compatibility evaluations against periodontopathogens and dental pulp stem cells, respectively.
2
Materials and methods
2.1
Materials
Poly(ɛ-caprolactone) pellets (PCL, M w = 80,000), gelatine type B from bovine skin (∼225 bloom, M w = 50,000), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP), and zinc oxide nanoparticles (ZnO, <100 nm particle size) were all purchased from Sigma–Aldrich (St. Louis, MO, USA) and used as-received. Phosphate-buffered saline solution (PBS, 1%) was prepared by mixing PBS tablets (MP Biomedicals, Santa Ana, CA, USA) and distilled water. The plastic syringes (5 mL) and 27G dispensing needles were obtained from Becton, Dickson and Company (Franklin Lakes, NJ, USA) and CML Supply (Lexington, KY, USA), respectively.
2.2
Synthesis of PCL and PCL/Gelatin membranes loaded with ZnO nanoparticles
The electrospinning system used in this work consisted of a high-voltage source (ES50P-10W/DAM, Gamma High-Voltage Research Inc., Ormond Beach, FL, USA), a syringe pump (Legato 200, KD Scientific Apparatus, Holliston, MA, USA), and a grounded stainless steel collecting drum connected to a high-speed mechanical stirrer (BDC6015, Caframo, Wiarton, ON, Canada), and was described in detail elsewhere . Processing parameters, including solution concentration, field strength, and flow rate were optimized for the distinct polymer systems to obtain defect-free fibrous membranes. Two stock polymer solutions were prepared in HFP; namely, pure PCL and a PCL/gelatin blend (PCL/GEL, ratio of 1:1, w/w). Briefly, the polymers were dissolved in HFP to produce a 10 wt.% solution (100 mg mL −1 ) and were stirred overnight. Next, ZnO nanoparticles were added at different concentrations (0, 5, 15, and 30 wt.%, relative to the total polymer weight); the mixtures were stirred for 24 h and then sonicated for 90 min before electrospinning to improve nanoparticle dispersion . Each solution was then loaded into the plastic syringe fitted with the 27G metallic blunt-tip and electrospun using the following parameters: rotating mandrel (120 rpm of speed), fixed spinning distance of 18 cm, flow rate of 1.5 mL/h, and different electric voltages, depending on the solution, as determined by an optimization process that involved experiments conducted on each variable. The obtained mats (hereafter referred to as membranes), i.e. pure PCL, PCL/GEL, and the ones containing ZnO nanoparticles, were collected at room temperature (RT) and then dried under vacuum for at least 48 h to complete removal of any remaining solvent .
2.3
Morphological (SEM/TEM) and chemical (EDS/FTIR) characterizations of the fibers
The electrospun membranes were observed under a field-emission scanning electron microscope (FE-SEM, Model JSM-6701F, JEOL, Tokyo, Japan) to evaluate the morphology and overall fiber architecture. In brief, samples taken from the different electrospun membranes were mounted on an Al stub and sputter-coated with Au-Pd prior to imaging. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to calculate the diameter of 50 single-fibers per image obtained from the FE-SEM analyses (3 images/group) at the same magnification (5000×); the fiber diameter was then averaged and reported as mean ± SD . Energy dispersive X-ray spectroscopy (EDX) was performed under FE-SEM to semi-quantitatively analyse the chemical composition of the fibers. Transmission electron microscopy (TEM, Model JEM-2010, JEOL) was also carried out to investigate the ZnO nanoparticles’ incorporation into the polymer fibers. Fourier transform infrared spectroscopy (FTIR) was done in the attenuated total reflection mode for the PCL, PCL/GEL, and membranes containing ZnO nanoparticles (ATR/FTIR-4100, JASCO, Easton, MD, USA), over the range of 700–4000 cm −1 at a resolution of 4 cm −1 to investigate the chemical characteristics of the membranes and ZnO incorporation .
2.4
Contact angle (CA)
The PCL and PCL/GEL solutions loaded or not with the distinct concentration of ZnO were electrospun into fibers using the aforementioned electrospinning parameters over microscope glass coverslips (Fisherbrand, Fisher Scientific, Loughborough, UK) mounted on the rotating mandrel ( n = 10). Next, the surface CA of the membranes was measured using a goniometer (Model PG-2, Gardco, Pompano Beach, FL, USA) by dropping three consecutive drops of distilled water (∼5 μL) per sample. The measured angles were then averaged.
2.5
Mechanical properties
The mechanical properties (i.e. tensile strength, Young’s modulus, and elongation at break) of all the synthesized electrospun membranes were evaluated by uni-axial tensile testing (expert 5601, ADMET, Norwood, MA, USA) . Rectangular samples (15 mm × 3 mm) were tested ( n = 8) under both dry (immediate testing without storage) or wet (tested after storage in PBS solution for 24 h) conditions at a crosshead speed of 1 mm min −1 . The specimen thickness was determined by measuring with calipers at three locations. Mechanical data were obtained from the stress-strain curves of each sample and expressed in MPa. The results are reported as mean ± standard deviation (SD).
2.6
Antimicrobial activity
The antibacterial activity of the processed fibrous membranes was evaluated against known periodontopathogens, i.e. Porphyromonas gingivalis ( Pg ) (ATCC 33277) and Fusobacterium nucleatum ( Fn ) (ATCC 25586) using agar diffusion assays ( n = 3/group/bacteria) . Bacteria were cultivated in Brain Heart Infusion (BHI) broth supplemented with 5 g yeast extract/L and 5% (v/v) vitamin K+ hemin (BHI-YE; Becton, Dickinson and Company) at 37 °C in an anaerobic GasPak jar for 24 h . Before testing, the minimum inhibitory concentration (MIC) of ZnO was determined using suspensions of ZnO nanoparticles in PBS at the following concentrations: 100, 250, 500, 1,000, 2,500, 5,000, and 10,000 μg/mL. In brief, 10 μL of each suspension was dropped on cultured blood agar plates containing the bacterial lawns, and the inhibition zones (in mm) were measured after 5 days of incubation . For the antibacterial activity of the membranes, disk-shaped samples (5-mm in diameter) were prepared and sterilized through ultraviolet (UV) irradiation for 1 h (30 min each side), before placing on the cultured blood agar plates as previously indicated . The inhibition zone (in mm) of each sample was measured after 5 days of incubation.
2.7
Cytotoxicity test
The guidelines provided by the International Standards Organization/ISO were followed in order to detect toxicity levels of the electrospun membranes . Human dental pulp stem cells (hDPSCs, AllCells LLC, Alameda, CA, USA) obtained from permanent third molars were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone Laboratories Inc., Logan, UT, USA) and 1% penicillin–streptomycin (Sigma) in a humidifier incubator at 37 °C, with 5% CO 2 . All the processed membranes were carefully cut into squares (15 mm × 15 mm) and sterilized under UV light (30 min each side) and by soaking in 70% ethanol for 30 min, followed by washing (5 min) twice in sterile PBS before testing . Next, sterile samples (surface ratio = 1 cm 2 /mL of medium) were incubated in DMEM for 48 h at 37 °C under a 5% CO 2 humidified atmosphere in order to produce extracts from each membrane . Extracts of sterile ultra-high molecular weight polyethylene (UHMWPE) were similarly obtained (negative control). Supernatants were then filtered through a membrane (Millipore ® ), and serial dilutions (100, 50, 25, 12.5, and 6.25 vol.%) were prepared from the extracts . Dilutions were also performed for the positive control (i.e. 0.3 vol.% phenol solution) . hDPSCs at passage 9 were seeded at a density of 3 × 10 3 /well and allowed to adhere in the wells of 96-well tissue culture microtiter plates. The media was replaced after 4 h by the corresponding extract concentrations, and negative and positive controls, which were dispensed into each well (100 μL) . Control columns of four wells were prepared with a medium without cells (blank), and a medium with cells but without the extract (100% survival) . The microplate was then incubated again and after 48 h, 20 μL of CellTiter 96 AQueous One Solution Reagent (Promega Corporation, Madison, WI, USA) was added to the test wells and allowed to react for 2 h at 37 °C in humidified 5% CO 2 atmosphere. Incorporated dye was measured by reading the absorbance at 490 nm in a microplate reader against a blank column .
2.8
Statistical analysis
The obtained data were statistically analysed (SigmaPlot version 12, Systat Software Inc., San Jose, CA, USA) using Analysis of Variance (One-Way for cytocompatibility test and Two-Way for all the other tests) and Tukey’s test for multiple comparison ( α = 5%).
2
Materials and methods
2.1
Materials
Poly(ɛ-caprolactone) pellets (PCL, M w = 80,000), gelatine type B from bovine skin (∼225 bloom, M w = 50,000), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP), and zinc oxide nanoparticles (ZnO, <100 nm particle size) were all purchased from Sigma–Aldrich (St. Louis, MO, USA) and used as-received. Phosphate-buffered saline solution (PBS, 1%) was prepared by mixing PBS tablets (MP Biomedicals, Santa Ana, CA, USA) and distilled water. The plastic syringes (5 mL) and 27G dispensing needles were obtained from Becton, Dickson and Company (Franklin Lakes, NJ, USA) and CML Supply (Lexington, KY, USA), respectively.
2.2
Synthesis of PCL and PCL/Gelatin membranes loaded with ZnO nanoparticles
The electrospinning system used in this work consisted of a high-voltage source (ES50P-10W/DAM, Gamma High-Voltage Research Inc., Ormond Beach, FL, USA), a syringe pump (Legato 200, KD Scientific Apparatus, Holliston, MA, USA), and a grounded stainless steel collecting drum connected to a high-speed mechanical stirrer (BDC6015, Caframo, Wiarton, ON, Canada), and was described in detail elsewhere . Processing parameters, including solution concentration, field strength, and flow rate were optimized for the distinct polymer systems to obtain defect-free fibrous membranes. Two stock polymer solutions were prepared in HFP; namely, pure PCL and a PCL/gelatin blend (PCL/GEL, ratio of 1:1, w/w). Briefly, the polymers were dissolved in HFP to produce a 10 wt.% solution (100 mg mL −1 ) and were stirred overnight. Next, ZnO nanoparticles were added at different concentrations (0, 5, 15, and 30 wt.%, relative to the total polymer weight); the mixtures were stirred for 24 h and then sonicated for 90 min before electrospinning to improve nanoparticle dispersion . Each solution was then loaded into the plastic syringe fitted with the 27G metallic blunt-tip and electrospun using the following parameters: rotating mandrel (120 rpm of speed), fixed spinning distance of 18 cm, flow rate of 1.5 mL/h, and different electric voltages, depending on the solution, as determined by an optimization process that involved experiments conducted on each variable. The obtained mats (hereafter referred to as membranes), i.e. pure PCL, PCL/GEL, and the ones containing ZnO nanoparticles, were collected at room temperature (RT) and then dried under vacuum for at least 48 h to complete removal of any remaining solvent .
2.3
Morphological (SEM/TEM) and chemical (EDS/FTIR) characterizations of the fibers
The electrospun membranes were observed under a field-emission scanning electron microscope (FE-SEM, Model JSM-6701F, JEOL, Tokyo, Japan) to evaluate the morphology and overall fiber architecture. In brief, samples taken from the different electrospun membranes were mounted on an Al stub and sputter-coated with Au-Pd prior to imaging. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to calculate the diameter of 50 single-fibers per image obtained from the FE-SEM analyses (3 images/group) at the same magnification (5000×); the fiber diameter was then averaged and reported as mean ± SD . Energy dispersive X-ray spectroscopy (EDX) was performed under FE-SEM to semi-quantitatively analyse the chemical composition of the fibers. Transmission electron microscopy (TEM, Model JEM-2010, JEOL) was also carried out to investigate the ZnO nanoparticles’ incorporation into the polymer fibers. Fourier transform infrared spectroscopy (FTIR) was done in the attenuated total reflection mode for the PCL, PCL/GEL, and membranes containing ZnO nanoparticles (ATR/FTIR-4100, JASCO, Easton, MD, USA), over the range of 700–4000 cm −1 at a resolution of 4 cm −1 to investigate the chemical characteristics of the membranes and ZnO incorporation .
2.4
Contact angle (CA)
The PCL and PCL/GEL solutions loaded or not with the distinct concentration of ZnO were electrospun into fibers using the aforementioned electrospinning parameters over microscope glass coverslips (Fisherbrand, Fisher Scientific, Loughborough, UK) mounted on the rotating mandrel ( n = 10). Next, the surface CA of the membranes was measured using a goniometer (Model PG-2, Gardco, Pompano Beach, FL, USA) by dropping three consecutive drops of distilled water (∼5 μL) per sample. The measured angles were then averaged.
2.5
Mechanical properties
The mechanical properties (i.e. tensile strength, Young’s modulus, and elongation at break) of all the synthesized electrospun membranes were evaluated by uni-axial tensile testing (expert 5601, ADMET, Norwood, MA, USA) . Rectangular samples (15 mm × 3 mm) were tested ( n = 8) under both dry (immediate testing without storage) or wet (tested after storage in PBS solution for 24 h) conditions at a crosshead speed of 1 mm min −1 . The specimen thickness was determined by measuring with calipers at three locations. Mechanical data were obtained from the stress-strain curves of each sample and expressed in MPa. The results are reported as mean ± standard deviation (SD).
2.6
Antimicrobial activity
The antibacterial activity of the processed fibrous membranes was evaluated against known periodontopathogens, i.e. Porphyromonas gingivalis ( Pg ) (ATCC 33277) and Fusobacterium nucleatum ( Fn ) (ATCC 25586) using agar diffusion assays ( n = 3/group/bacteria) . Bacteria were cultivated in Brain Heart Infusion (BHI) broth supplemented with 5 g yeast extract/L and 5% (v/v) vitamin K+ hemin (BHI-YE; Becton, Dickinson and Company) at 37 °C in an anaerobic GasPak jar for 24 h . Before testing, the minimum inhibitory concentration (MIC) of ZnO was determined using suspensions of ZnO nanoparticles in PBS at the following concentrations: 100, 250, 500, 1,000, 2,500, 5,000, and 10,000 μg/mL. In brief, 10 μL of each suspension was dropped on cultured blood agar plates containing the bacterial lawns, and the inhibition zones (in mm) were measured after 5 days of incubation . For the antibacterial activity of the membranes, disk-shaped samples (5-mm in diameter) were prepared and sterilized through ultraviolet (UV) irradiation for 1 h (30 min each side), before placing on the cultured blood agar plates as previously indicated . The inhibition zone (in mm) of each sample was measured after 5 days of incubation.
2.7
Cytotoxicity test
The guidelines provided by the International Standards Organization/ISO were followed in order to detect toxicity levels of the electrospun membranes . Human dental pulp stem cells (hDPSCs, AllCells LLC, Alameda, CA, USA) obtained from permanent third molars were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone Laboratories Inc., Logan, UT, USA) and 1% penicillin–streptomycin (Sigma) in a humidifier incubator at 37 °C, with 5% CO 2 . All the processed membranes were carefully cut into squares (15 mm × 15 mm) and sterilized under UV light (30 min each side) and by soaking in 70% ethanol for 30 min, followed by washing (5 min) twice in sterile PBS before testing . Next, sterile samples (surface ratio = 1 cm 2 /mL of medium) were incubated in DMEM for 48 h at 37 °C under a 5% CO 2 humidified atmosphere in order to produce extracts from each membrane . Extracts of sterile ultra-high molecular weight polyethylene (UHMWPE) were similarly obtained (negative control). Supernatants were then filtered through a membrane (Millipore ® ), and serial dilutions (100, 50, 25, 12.5, and 6.25 vol.%) were prepared from the extracts . Dilutions were also performed for the positive control (i.e. 0.3 vol.% phenol solution) . hDPSCs at passage 9 were seeded at a density of 3 × 10 3 /well and allowed to adhere in the wells of 96-well tissue culture microtiter plates. The media was replaced after 4 h by the corresponding extract concentrations, and negative and positive controls, which were dispensed into each well (100 μL) . Control columns of four wells were prepared with a medium without cells (blank), and a medium with cells but without the extract (100% survival) . The microplate was then incubated again and after 48 h, 20 μL of CellTiter 96 AQueous One Solution Reagent (Promega Corporation, Madison, WI, USA) was added to the test wells and allowed to react for 2 h at 37 °C in humidified 5% CO 2 atmosphere. Incorporated dye was measured by reading the absorbance at 490 nm in a microplate reader against a blank column .
2.8
Statistical analysis
The obtained data were statistically analysed (SigmaPlot version 12, Systat Software Inc., San Jose, CA, USA) using Analysis of Variance (One-Way for cytocompatibility test and Two-Way for all the other tests) and Tukey’s test for multiple comparison ( α = 5%).
3
Results
3.1
Morphological and chemical characterizations of the fibers
The PCL- and PCL/GEL-based membranes presented, respectively, a heterogeneous (from 93 to 2,223 nm) and homogeneous (from 56 to 1184 nm) fiber diameter distribution ( Fig. 1 a). The presence of gelatine produced thicker fibers compared to those gelatine-free, although only in groups containing none or low content (5 wt.%) of ZnO. The incorporation of higher amounts of ZnO (higher than 15 wt.%) resulted in rough fibers with distinct shape (e.g., swollen areas) and morphology, although the PCL membranes were more negatively affected than those constituted of gelatine ( Fig. 2 ). As shown in the EDS analysis ( Fig. 1 b), ZnO nanoparticles were successfully incorporated into the membranes. The nanoparticles differed in shape, although most of them were rod-like and nano-sized (i.e. below 100 nm, with an average size of 65 nm) ( Fig. 3 ). While the neat fibers were visually smooth ( Fig. 4 a), the nanocomposite fibers presented some swollen areas and several areas embedded with ZnO nanoparticles ( Fig. 4 b). Lastly, the FTIR analysis also revealed incorporation of ZnO into the membranes ( Fig. 5 ) and gelatine into the PCL/GEL-based membranes ( Fig. 5 b). The presence of the peak located at 2357 cm −1 could be observed in the ZnO powder and also in the ZnO-incorporated membranes, but not in the neat membranes. FTIR spectra showed some common peaks for all membranes located at 2944–2861 and 1721 cm −1 , which corresponded, respectively, to the presence of the C–H bond of saturated carbons and the ester-carbonyl group (–CO stretching) found in PCL . In addition, three characteristic peaks confirmed the incorporation of gelatine into the PCL/GEL membranes: one located at 3297 cm −1 (amide-A), another at 1627 cm −1 (amide-I), and the last at 1524 cm −1 (amide-II).
