Nitric Oxide-Releasing Nanofibrous Scaffolds Based on Silk Fibroin and Zein with Enhanced Biodegradability and Antibacterial Properties

Abstract

Clinical applications of scaffolds and implants have been associated with bacterial infection resulting in impaired tissue regeneration. Nanofibers provide a versatile structure for both antimicrobial molecule delivery and tissue engineering. In this study, the nitric oxide (NO) donor molecule S-nitrosoglutathione (GSNO) and the natural biodegradable polymer zein (ZN) were combined with silk fibroin (SF) to develop antibacterial and biodegradable nanofibrous scaffolds for tissue engineering applications. The compatibility and intermolecular interactions of SF and ZN were studied using differential scanning calorimetry and Fourier transform infrared spectroscopy. The incorporation of ZN increased the hydrophobicity of the fibers and resulted in a more controlled and prolonged NO release profile lasting for 48 h. Moreover, the degradation kinetics of the fibers was significantly improved after blending with ZN. The results of tensile testing indicated that the addition of ZN and GSNO had a positive effect on the strength and stretchability of SF fibers and did not adversely affect their mechanical properties. Finally, due to the antibacterial properties of both NO and ZN, the SF–ZN–GSNO fibers showed a synergistically high antibacterial efficacy with 91.6 ± 2.5% and 77.5 ± 3.1% reduction in viability of adhered Staphylococcus aureus and Escherichia coli after 24 h exposure, respectively. The developed NO-releasing fibers were not only antibacterial but also non-cytotoxic and successfully enhanced the proliferation and growth of fibroblast cells, which was quantitatively studied by a CCK-8 assay and visually observed through fluorescent staining. Overall, SF–ZN–GSNO fibers developed in this study were biodegradable and highly antibacterial and showed great cytocompatibility with fibroblasts, indicating their promising potential for a range of tissue engineering and medical device applications.

Graphical Abstract

1. INTRODUCTION

Biodegradable polymeric scaffolds play a critical role in regenerative medicine by providing temporary structural support for cells to reside in and reconstruct the damaged tissue. For efficient cell binding, proliferation, and neo tissue formation, scaffolds must be biocompatible and of close similarity to the native extracellular matrix (ECM).¹ Electrospinning is a popular, low-cost, and versatile technique for fabricating micro/nanofibrous scaffolds that can mimic the fibrillar structure of the ECM. Electrospun fibers offer many advantages for tissue engineering including having a porous network, a high specific surface area favorable for cell attachment, and allowing for efficient diffusion of nutrients and oxygen.² These fibers can also be incorporated with different drugs, therapeutics, and antibacterial agents for specific applications.

Nitric oxide (NO) donor molecules have recently found increasing interest to be incorporated into polymeric scaffolds for wound healing and tissue engineering applications.^3,4 NO is a gaseous molecule endogenously produced by NO synthase enzymes in the pico-nano molar range and plays important roles in the wound healing process by regulating the inflammatory response, enhancing cell proliferation, collagen formation, and angiogenesis.⁵ In addition, NO is part of the innate immune system and can effectively neutralize infectious bacteria and viruses when released from macrophages and other immune cells.⁶

Scaffolds can get infected by opportunistic bacteria at the time of implantation or later by the spread of infection from other parts of the body, leading to serious consequences such as tissue damage, a prolonged healing time, and patient morbidity and mortality.⁷ The adherence of bacteria to the scaffolds may lead to the formation of biofilms on the surface, rendering treatment with antibiotics barely effective. Biofilms are multicellular communities of bacteria that have encased themselves in a matrix composed of extracellular polymeric substances (EPSs) and are highly impermeable to antibacterial agents. NO has been previously shown to have the ability to disperse biofilms into more vulnerable planktonic state bacteria by decreasing the levels of the c-di-GMP messenger molecule that is responsible for regulating the biofilm integrity.⁸ Therefore, due to its strong antibacterial activity, biofilm dispersal ability, and multifaceted roles in regulating the wound healing process, NO is an ideal candidate to be incorporated into scaffolds to prevent infection and promote tissue regeneration.

In the past, NO donor molecules such as diazeniumdiolates (NONOates) and S-nitrosothiols (RSNOs) have been incorporated into different polymeric fibers for tissue engineering, wound healing, and other biomedical applications.^9–13 The previously developed NO-releasing fibers were mostly made of synthetic polymers to avoid limitations frequently associated with the use of hydrophilic natural polymers such as high leaching of the donor molecule, burst NO release kinetics, and poor mechanical properties. However, despite their disadvantages, biopolymers are of greater popularity for tissue engineering applications owing to their lower immunogenicity, higher biocompatibility, biodegradability, and biological recognition compared to synthetic polymers.^14,15 Therefore, further research is required into designing biodegradable and biocompatible scaffolds based on naturally derived polymers with improved NO release kinetics and good mechanical properties.

Silk fibroin (SF) is a protein that can be obtained from the cocoons of the silkworm. SF is widely used in biomedical applications due to its outstanding properties such as high mechanical properties, excellent biocompatibility, water-based processing, and ability to be shaped into different material formats.^16,17 When in a liquid format, SF macromolecules are randomly dispersed bearing helical or random coil conformations.¹⁸ However, for most applications, SF is treated either physically (i.e., sonication, water vapor annealing) or chemically (i.e., methanol, acetone, or ethanol treatment) to induce a conformational transition in its structure into crystalline β-sheets that make SF non-soluble and mechanically strong but also highly resistant to degradation.¹⁹ Crystallized SF has been shown to have a very slow degradation rate in aqueous media.²⁰ As tissue engineering constructs are required to be degraded gradually to allow the regenerating tissue to be replaced with them over time, it is important to enhance the degradability of SF-based scaffolds. One simple approach to improve the degradation kinetics of SF for tissue engineering applications is to blend it with other more biodegradable polymers.^21,22

Zein (ZN) is a naturally derived protein obtained from corn that has been widely used for wound healing and scaffold applications due to its biodegradability and biocompatibility.²³ However, ZN suffers from poor mechanical properties, and therefore, for most biomedical applications, it needs to be combined with other more mechanically robust polymers or filler particles to enhance the mechanical properties.²⁴ Using a single-jet electrospinning method, in this study, we fabricated composite nanofibers based on SF and ZN to develop mechanically strong yet biodegradable scaffolds.

To endow the scaffolds with antibacterial activity and bioactivity, a NO donor molecule, S-nitrosoglutathione (GSNO), was embedded into these fibers. GSNO is an endogenous RSNO, naturally available in vivo, that is known for its great biocompatibility and excellent stability among other RSNOs.^25,26 The NO-releasing SF–ZN scaffolds (SF–ZN–GSNO) were observed under scanning electron microscopy (SEM) to evaluate their morphology and size distribution. The long-term enzymatic degradability of the fibers along with their wettability and mechanical properties under uniaxial tensile loading were tested. In addition, the cumulative leaching of GSNO from the fibers and their NO release kinetics were studied. Finally, antibacterial activity of the scaffolds against Gram-positive and Gram-negative bacteria and their cytocompatibility with 3T3 fibroblast cells were also investigated.

2. MATERIALS AND METHODS

2.1. Materials.

Bombyx mori (B. mori) silk cocoons were obtained from Paradise Fibers (USA). ZN from maize (Z3625), DAPI, hydrochloric acid, sodium nitrite, methanol (≥99.8%), lithium bromide, Cell Counting Kit-8 (CCK-8), and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich (St. Louis, MO). Trypsin (1:250) for the degradation test was purchased from GoldBio. Acetone was purchased from VWR (Radnor, PA). l-glutathione (reduced 98+%) was obtained from Alfa Aesar (Ward Hill, MA). Spectra/Por 3 regenerated cellulose dialysis membrane tubings (3.5 kDa molecular weight cutoff) and Alexa Fluor 488 Phalloidin were purchased from Thermo Fisher Scientific (Waltham, MA). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin–EDTA were purchased from Corning (Manassas, VA). The penicillin–streptomycin (Pen–Strep) antibiotic and fetal bovine serum (FBS) were bought from Gibco-Life Technologies (Grand Island NY 14072). LB broth was obtained from Fisher Bioreagents. LB Agar was obtained from Difco Laboratories. The bacterial strains Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) and 3T3 mouse fibroblast cells (ATCC 1658) were purchased from American Type Culture Collection (ATCC).

2.2. GSNO Synthesis.

GSNO was synthesized following a previously established protocol with slight modification.²⁷ First, glutathione (2.93 mmol, 900 mg) was dissolved in 4 mL of deionized (DI) water combined with 1.25 mL of 2 M HCl. The solution was allowed to chill in an ice bath before the addition of NaNO₂ (3 mmol, 207 mg) for nitrosation. Afterward, the solution was allowed to chill in the ice bath for an additional 40 min, and then, 5 mL of chilled acetone was added to it and stirred for 10 min. The formed dark-pink GNSO precipitates were collected by vacuum filtration, rinsed with cold DI water and acetone, and allowed to dry in a vacuum desiccator overnight.

2.3. SF Extraction.

Aqueous SF solution was extracted from B. Mori silkworm cocoons according to a previously described procedure.²⁸ Briefly, the cocoons were first degummed by boiling in 20 mM sodium carbonate solution for 30 min. The degummed cocoons were rinsed thoroughly with DI water and allowed to dry at room temperature. The dried SF was then dissolved in 9.3 M lithium bromide at 60 °C for 4 h and dialyzed against DI water in a dialysis membrane (3.5 kDa cutoff) for 48 h. The obtained aqueous SF solution was then filtered to remove any contaminants and lyophilized to obtain SF sponges.

2.4. Fabrication of Fibers.

The fibers were prepared by a single-jet electrospinning method. The prepared SF sponge was first dissolved in HFIP to obtain a 10% w/v SF solution. The solution was then transferred into a plastic 3 mL syringe with an 18-gauge stainless steel blunt-tip needle and mounted on a custom-made electrospinning setup. The needle tip was set at a 10 cm distance from a grounded collector, and the fibers were spun at a flow rate of 1 mL/h with an applied voltage of 14 kV. The composite fibers were made from 10% w/v solutions containing SF and ZN at a ratio of 2:1, respectively. To fabricate the NO-releasing fibers, 15% w/w GSNO was added to the electrospinning solutions with respect to polymer weight and sonicated to obtain a homogeneous mixture. The GSNO content of the fibers was determined based on previous studies done by our group and to ensure good electrospinability of the polymeric solutions.^12,13 The as-spun scaffolds were finally immersed in methanol for 5 min to induce β-sheet formation in SF and were placed in a vacuum desiccator to dry at room temperature for 24 h.

2.5. Differential Scanning Calorimetry.

The miscibility and compatibility of SF and ZN polymers were studied using a differential scanning calorimeter (TA Instruments-DSC 250, DE, USA). Untreated SF, ZN, and SF–ZN blend films were prepared using 10% w/v polymer solutions in HFIP (similar to the polymer solutions prepared for the electrospinning process). The films were prepared by a solvent casting method and drying at room temperature overnight to use for the experiment. Pre-determined weights of samples were completely sealed in aluminum pans and heated at a constant rate of 10 °C/min over a temperature range of 25–250 °C. Nitrogen gas was purged at a rate of 50 mL/min to ensure maintaining an inert atmosphere.

2.6. SEM Imaging.

The morphology of the fibers was observed using SEM (FEI Teneo, FEI Co). The fibers were sputter-coated with a 10 nm thick gold layer prior to imaging. The images were taken at an excitation voltage of 5.00 kV. The average fiber diameter was measured using ImageJ software by analyzing at least 50 random fibers in different SEM images.

2.7. Fourier Transform Infrared Spectroscopy and Deconvolution Analysis.

The Fourier transform infrared (FTIR) spectra of the nanofibrous mats were recorded using an attenuated total reflection method using a Nicolet 6700 Spectrometer (Thermo Electron Corporation, MA, USA). All spectra were obtained in the range of 900–3700 cm⁻¹ with a resolution of 4 cm⁻¹ and a total of 128 scans per measurement. At least three spectra were recorded for each sample type. The deconvolution analysis of the amide I band for SF and SF–ZN nanofibrous mats was performed using OriginPro 8 software as previously described.^18,19,29,30 The second-derivative plot of each spectrum was used to locate the underlying peaks, and the fitting was performed using a Gaussian model with an equal fixed width for all the considered peaks. The crystallinity content was calculated by measuring the percentage ratio of the area of β-sheet peaks to the total area of the amide I peak.

2.8. In Vitro Biodegradation.

To evaluate the enzyme-mediated degradation of the scaffolds, known weights of the fibers were submerged in 1 mg/mL trypsin (1:250) solution in PBS and incubated at 37 °C. At different time points, the fibers were taken out, rinsed with DI water, and freeze-dried. The percentage of difference in weight of the dried scaffolds before and after enzymatic degradation was calculated and plotted against time to obtain the degradation profiles.

2.9. Tensile Testing.

The mechanical properties of the nanofibrous scaffolds were investigated using a universal testing machine (Mark-10 ESM303) according to ASTM D-882. The fibers were cut into 50 mm long and 5 mm wide T-bar strips, fixed between the grips, and stretched at a rate of 1 mm min⁻¹. Before each measurement, the thickness of the sample was measured to calculate the cross-sectional area that the force was applied to. Stress and strain values were calculated using below equations

$$\text{stress (MPa)}=\frac{\text{applied force (N)}}{{\text{cross sectional area (mm}}^{\text{2}}\text{)}}$$

$$\text{strain (\%)}=\frac{\text{change in original length (mm)}}{\text{original length (mm)}}\times 100$$

The Young’s modulus, tensile strength, and elongation at break (EB) for each specimen were measured according to the obtained stress/strain profiles.

2.10. Contact Angle Measurement.

Static contact angle measurements were carried out using an Ossila contact angle goniometer (Sheffield, UK). 5 μl of DI water was dropped onto flat films of each sample type secured on a glass slide, and then, contact angles for each sample were recorded from the frame captures using the sessile drop method.

2.11. NO Release Measurement.

NO release from GSNO-loaded fibers was measured via chemiluminescence using a Sievers nitric oxide analyzer (NOA) model 280i (Boulder, CO). Chemiluminescence NOA instruments offer a wide dynamic linear range (typically 0.5 ppb to 500 ppm NO) with a detection limit of ~0.5 parts per billion for gas-phase NO by volume and ~1 pmol for the measurement of NO and its reaction products in liquid samples. The fibrous scaffolds were weighed and submerged in 0.01 M PBS containing EDTA at 37 °C in an amber reaction vessel to protect from light. A continuous flow of nitrogen gas at a rate of 200 mL min⁻¹ carried the NO released from the sample to the NOA system. The NO levels at each time point were recorded in ppb units and converted to a standard NO release rate (×10⁻¹⁰ mol mg⁻¹ min⁻¹) using the NOA instrument constant.

2.12. GSNO Leaching.

The leaching of NO donor, GSNO, from the SF–GSNO and SF–ZN–GSNO fibers was measured by UV–vis spectroscopy. Fibers were weighed and incubated in a known volume of PBS (pH 7.4) at 37 °C. At different time points, the absorbance was recorded at 340 nm, which is the S–NO bond characteristic peak present in RSNOs. A GSNO calibration curve in PBS was used to determine the concentration of the leached GSNO from the fibers. The percent of GSNO leached at each time point was calculated by dividing the cumulative amount of GSNO leached out to the initial amount of GSNO loaded into the fibers. Both SF–GSNO and SF–ZN–GSNO had a similar GSNO content of 15% w/w with respect to the polymer weight.

2.13. Evaluation of the Antibacterial Activity.

To determine the antimicrobial efficacy, synthesized fibers and their controls were exposed to S. aureus and E. coli for 24 h, and the viability of adhered bacteria was quantified. First, isolated S. aureus and E. coli were separately inoculated in LB media at 150 rpm at 37 °C for 12–14 h. The resulting liquid culture was centrifuged at 2500 rpm for 7.5 min, rinsed with sterile PBS, and centrifuged again at 2500 rpm for 7.5 min. The pellets were subsequently resuspended in sterile PBS to reach a final bacterial concentration of ~10⁸ cfu mL⁻¹. In a 24-well plate, individual samples (SF, SF–GSNO, SF–ZN, and SF–ZN–GSNO) were placed in separate wells. Samples were massed prior to exposure. The well plates were then incubated at 37 °C at 150 rpm for 24 h. After the 24 h exposure period, samples were removed, rinsed with sterile PBS, and homogenized for 60 s at 25,000 rpm in a 15 mL falcon tube containing 1 mL of PBS to detach adhered bacteria. The resulting bacterial solution was serially diluted and plated on LB agar plates, and the agar plates were incubated at 37 °C overnight. Colony-forming units were quantified to determine the number of viable bacteria per mg of each sample, and the percentage reduction with respect to control samples according to the following equation (where C = cfu mg⁻¹)

$$\text{\% reduction in adhered bacterial viabilty}=\frac{C_{\text{ctrl}}-C_{\text{test}}}{C_{\text{ctrl}}}\times 100$$

2.14. Cell Viability and Morphology Assessment.

An in vitro CCK-8 assay was performed to evaluate the potential cytotoxicity of the scaffolds toward mammalian cells. Briefly, cell culture was carried out by seeding 3T3 mouse fibroblast cells (ATCC 1658) in a 75 cm² T-flask containing complete DMEM with 10% FBS and 1% Pen–Strep. The flask with cells was incubated in a humidified environment at 37 °C with 5% CO₂. After reaching >80% confluency, cells were detached from the flask using 0.18% trypsin and 5 mM EDTA, stained with Trypan blue, and counted using a hemocytometer. 100 μL of the diluted cell suspension was then transferred to each well of a 96-well plate, with a density of 5000 cells per well, and allowed to form a monolayer overnight. Sample leachates were prepared by soaking the fibers in DMEM at a concentration of 10 mg/mL and incubating at 37 °C for 24 h. The obtained leachates were then replaced with the media in each well and exposed to cells for another 24 h. The effect of the leachates on cell viability was determined using the CCK-8 cell counting kit based on the manufacturer’s protocol (Sigma-Aldrich). According to the protocol, 10 μL of a highly water-soluble tetrazolium salt, WST-8, was added to each of the wells and incubated for 2 h. WST-8 is reduced by the dehydrogenases in the living cells to produce an orange-colored formazan dye that can be detected at 450 nm. After measuring the absorbance at 450 nm using a spectrophotometer microplate reader, the relative cell viability was calculated using the below equation. Cells maintained in DMEM with no leachates were used as controls (n = 6)

$$\text{Cell viability (\%)}=\frac{\text{absorbance (treated cells)}}{\text{absorbance (control cells)}}\times 100$$

The cytocompatibility of the scaffolds was further assessed by investigating the cell morphology and adhesion on the samples using fluorescence microscopy. The fibers were first electrospun onto glass slides and sterilized by UV radiation for 30 min. The fibroblast cells were then seeded on the fibers at a density of 15,000 cells/cm² and placed in a humidified incubator with 5% CO₂ at 37 °C for 48 h. To observe the cell morphology on the scaffolds, DAPI and Alexa Fluor-488 conjugated phalloidin dyes were used to stain the nuclei and cytoplasm, respectively. First, the cell-seeded scaffolds were rinsed with PBS, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.2% Triton-X-100 for another 10 min. Then, a 1:500 dilution of phalloidin in PBS was applied to cells for 20 min, followed by DAPI (300 nM in PBS) for 3 min. The stained cells were finally imaged using a fluorescent cell imaging system (AMG EVOS FL digital inverted microscope) and studied for overall attachment and growth.

2.15. Statistical Analysis.

A one-way analysis of variance was used to compare any significant difference. All data are reported as a mean ± standard deviation collected from three data points unless otherwise noted. A probability value (p) of less than 0.05 (p < 0.05) was considered to be statistically significant.

3. RESULTS AND DISCUSSION

3.1. SF and ZN Compatibility Study.

Differential scanning calorimetry (DSC) is the most widely used method for polymer compatibility analysis.^31,32 To investigate the miscibility and compatibility of SF and ZN polymers for fabrication of composite nanofibrous scaffolds, their glass-transition temperatures (T_g) before and after blending were studied using DSC. According to previous studies, the presence of a single T_g in the thermogram of the blended polymer composite is an indicator of a homogeneous and miscible polymer system. However, appearance of more than one T_g suggests the polymers with full or partial immiscibility.³¹ The DSC results shown in Figure 1 pointed out a T_g of 179.7 °C and an exothermic crystallization peak at 224.5 °C for untreated SF.^33,34 In addition, a glass transition at 156.4 °C was observed for amorphous ZN, consistent with the results of previous studies.^23,32 The SF–ZN polymer blend thermogram showed a single common glass transition at 148.9 °C, demonstrating the great miscibility and compatibility of SF and ZN and the formation of a one-phase polymer system.

Figure 1.

DSC profiles of untreated SF, ZN, and SF–ZN blend films.

3.2. Fiber Morphology and Size Analysis.

The fibers’ size and morphology were studied using SEM. Figure 2 shows the SEM images of different fibers and their corresponding size distribution graphs. The electrospun fibers all had a ribbon-shaped morphology that was attributed to the highly volatile nature of the HFIP solvent used. It has been demonstrated previously that fast and non-homogenous evaporation of the electrospinning solvent results in the formation of a shell on the cylindrical polymer jet, causing it to further collapse into a flat ribbon-shaped fiber due to atmospheric pressure.^35–38 SF fibers fabricated in this study had an average diameter of 1744.1 ± 553.6 nm. After the addition of GSNO, narrower size distribution and a slightly decreased fiber diameter (1135.2 ± 362.4 nm) were observed, although not significant (p > 0.05). SF–ZN and SF–ZN–GSNO fibers displayed significantly reduced fiber diameters of 491.5 ± 129.5 and 527.8 ± 119.5 nm (p < 0.001), respectively. Consistent with the results of other studies, the same effect has been seen previously, where the addition of ZN to polymeric electrospinning solutions such as polyvinyl alcohol,³⁹ dextran,⁴⁰ and gelatin⁴¹ caused a drop in the viscosity, favoring the formation of fibers with smaller diameters. In fact, thinner fibers with diameters in the nanoscale are desired for scaffold-related applications due to their enhanced cellular responses and closer similarity to the nanofibrous structure of the native ECM.^42,43 Therefore, SF–ZN–GSNO fibers can provide an ideal substrate for tissue growth and regeneration.

Figure 2.

SEM images of (A) SF, (B) SF–GSNO, (C) SF–ZN, and (D) SF–ZN–GSNO fibers and their corresponding diameter distribution graphs. Scale bars = 10 μm.

3.3. FTIR Spectroscopy and Amide I Band Deconvolution Analysis.

FTIR analysis was performed to study the intermolecular interactions between the components of the nanofibrous scaffolds. In the SF and SF–GSNO spectra (Figure 3), the characteristic protein peaks of amide I, amide II, and amide III bonds related to the stretching vibrations of C=O, C–N, and C–C/C–H were respectively observed at 1630, 1525, and 1234 cm⁻¹ wavelengths. Moreover, the peak appearing at 3200–3400 cm⁻¹ was attributed to the N–H and O–H stretching vibrations. The addition of GSNO did not cause any observable changes in the SF protein spectra. However, after addition of ZN, a sharp decrease in the intensity of the characteristic amide bond peaks and O–H/N–H stretching vibrations was noticed, indicating the formation of hydrogen bonding between ZN and SF proteins as described previously.⁴¹ Intermolecular hydrogen bonding between different compounds of a composite material is indicative of their compatibility and ensures the formation of a homogeneous single-phase polymer blend.

Figure 3.

FTIR spectra of SF, SF–GSNO, SF–ZN, and SF–ZN–GSNO nanofibrous scaffolds.

To further investigate the conformational changes occurring in SF after addition of ZN, deconvolution analysis of the amide I band was carried out, and the results are shown in Figure 4. As expected, methanol-treated SF fibers showed a high level of crystallinity with a β-sheet content of 41.6% (Figure 4A). The addition of ZN caused a shift in the intensity center of the amide I band to higher wavenumbers, suggesting a decrease in the crystallinity or β-sheet content of SF, which was measured to be 34.8% in the SF–ZN fibers (Figure 4B). According to previous studies and as corroborated by the FTIR spectra of the samples, this can be attributed to the partial breakage of the hydrogen bonds between SF macromolecules and the formation of new hydrogen bonds between SF and the amorphous ZN structure that leads to a lower crystallinity degree of SF in composite SF–ZN fibers compared to pure SF fibers.⁴⁴

Figure 4.

Deconvoluted amide I band of (A) SF and (B) SF–ZN nano fibrous scaffolds. β = β-sheets.

3.4. In Vitro Biodegradation.

Degradability is an essential feature for tissue engineering scaffolds. Scaffolds provide a temporary substrate for cells to adhere, grow, and repair or reconstruct the damaged tissue. For cells to be able to replace the scaffold over time, degradation should take place at an optimal rate, matching that of the new tissue being regenerated.⁴⁵ The degradation kinetics of the materials is mainly dictated by their structure. SF is composed of amorphous hydrophilic random coils and helical structures known as silk I as well as hydrophobic crystalline β-sheets known as silk II structures. Certain physical and chemical treatments such as sonication, water vapor annealing, methanol, and heat treatment can induce a conformational transition from silk I to silk II structures. The dominance of packed crystalline β-sheets in the SF structure makes it non-soluble and highly resistant to degradation in aqueous media.⁴⁶ In the current study, the accelerated enzyme-mediated degradation profiles of the fibers were obtained using trypsin (1:250) solution, and the results are depicted in Figure 5. Methanol-treated SF fibers had a very slow rate of degradation, losing only 19.6 ± 5.7% of their initial mass after 30 days in trypsin media. The addition of ZN to the SF electrospinning solutions caused a significant shift in the biodegradation kinetics of the fibers. ZN is a hydrophobic yet biodegradable polymer that has been increasingly explored for the development of scaffolds and implantable biomaterials in the past decades due to its biodegradability and biocompatibility.^47,48 Despite undergoing the same methanol treatment, SF–ZN fibers displayed a notably higher rate of degradation (p < 0.01), with 66.1 ± 1.5% of their initial weight being lost during the 30-day period. This is in part due to the faster degradation rate of ZN as an amorphous, naturally degradable polymer as well as the decreased crystallinity degree or β-sheet content of SF after addition of ZN which was demonstrated by FTIR deconvolution analysis of the amide I band in Figure 4. It was further observed that the presence of GSNO did not cause any significant differences in the degradation rates of the fibers. SF and ZN are naturally derived proteins, and their degradation products are biocompatible amino acids that can be metabolized by the body.⁴⁶ The composite SF–ZN–GSNO fibers prepared in this study showed enhanced biodegradation kinetics compared to pure SF and SF–GSNO fibers, which is desirable for scaffolding applications.

Figure 5.

Enzyme-mediated degradation profile of the fibrous scaffolds in trypsin (1:250) solution at 37 °C in the dark.

3.5. Mechanical Properties.

The mechanical behavior of biomaterials is of great importance in determining their stability and biological performance for different applications. SF has uniquely high mechanical properties among other natural polymers owing to its ability to undergo conformational transitions to form well-ordered crystalline β-sheets in its structure. In this study, uniaxial tensile testing was performed to investigate the effects of adding ZN and GSNO on the mechanical properties of the SF fibers. The results of the test are displayed in Figure 6 and summarized in Table 1. The SF fibers had an ultimate tensile strength (UTS) of 13.65 ± 0.99 MPa and an EB of 10.23 ± 0.87%. After the addition of GSNO, a significant increase in the UTS of SF fibers was observed (p < 0.05), which is consistent with the results of other studies blending NO donors into polymeric fibers.¹² However, no statistically significant difference was found between the EB of SF fibers with and without GSNO. Moreover, the composite SF–ZN fibers demonstrated a significantly enhanced EB (p < 0.05) that can be attributed to an increase in molecular entanglements between SF and ZN macromolecules, facilitating the slippage of polymer chains under stress as observed in previous studies.⁴¹ While the fibers showed different UTS and EB values, no significant changes in the Young’s modulus were found between any of the samples. Overall, the results indicated that the addition of ZN and GSNO improved the strength and stretchability of SF fibers and did not cause any adverse effects on their mechanical properties.

Figure 6.

Stress/strain diagram of SF, SF–GSNO, SF–ZN, and SF–ZN–GSNO nanofibrous scaffolds. The diagram is representative of the mechanical behavior of different samples under applied tensile stress obtained from three replicas.

Table 1.

Mechanical Properties of SF, SF–GSNO, SF–ZN, and SF–ZN–GSNO Fibers

samples	UTS (MPa)	EB (%)	Young’s modulus (MPa)
SF	13.65 ± 0.99	10.23 ± 0.87	2.40 ± 0.03
SF-GSNOxz	20.65 ± 4.05a	9.85 ± 1.41	2.43 ± 0.08
SF-ZN	18.61 ± 0.83	17.13 ± 3.96a	2.24 ± 0.08
SF-ZN-GSNO	17.26 ± 0.58	13.15 ± 1.83	2.36 ± 0.09

^aShows (p < 0.05) compared to SF fibers.

3.6. Surface Wettability.

The surface wettability of biomaterials is an important factor influencing their interactions with cells. A scaffold surface with moderate wettability is favorable for initial attachment and migration of cells, leading to enhanced healing rates.⁴⁹ The surface wettability of the samples was studied by measuring their water contact angle (WCA), and the results can be seen in Figure 7. To avoid the porous network of fibers affecting contact angle measurements, polymeric films of each sample were prepared and used for this study. SF films showed an average WCA of 56.3 ± 0.3°, consistent with the values reported by previous studies. ZN is known to be a hydrophobic protein composed of mainly non-polar amino acids. Therefore, after the addition of ZN, the WCA of SF films was significantly increased to 83.2 ± 2.3° (p < 0.001). However, the blending of GSNO caused a slight reduction in the WCA of both SF and SF–ZN films. SF–ZN–GSNO films showed moderate wettability with a WCA of 78.6 ± 0.9°, suggesting their suitability as scaffolds for cellular adhesion and growth.

Figure 7.

Static WCA of SF, SF–GSNO, SF–ZN, and SF–ZN–GSNO.

3.7. In Vitro NO Release Kinetics and GSNO Leaching.

In order to exert effective antibacterial and therapeutic activity, NO-releasing biomaterials with sustained release kinetics are demanded. Despite the increasing number of NO-releasing scaffolds and wound dressings being developed, only a few are reported to provide steady and prolonged NO release profiles lasting for more than 24 h. NO release from RSNOs such as GSNO is triggered by their exposure to heat, light, and metal ions with catalytic activity (e.g., Cu²⁺), resulting in the homolytic cleavage of the S–NO bond and the formation of NO and disulfide species according to the below equation⁵⁰

$$2\text{RSNO}\to \text{RSSR}+\text{2NO}$$

In this study, the release kinetics of NO from GSNO-blended fibers was tested at 37 °C in dark conditions using NOA, and the results are presented in Figure 8. SF fibers had an initial burst release of NO with an average rate of 1.39 ± 0.26 × 10⁻¹⁰ mol min⁻¹ mg⁻¹ in the first hour, which was gradually decreased to 0.09 ± 0.01 × 10⁻¹⁰ mol min⁻¹ mg⁻¹ at 24 h. After 24 h, the NO release rates from SF fibers were not detectable by NOA anymore. As can be seen in Figure 8, the burst release of NO from SF fibers during the first hour was notably mitigated in the SF–ZN–GSNO fibers (p < 0.01). The highest release rate (0.93 ± 0.26 × 10⁻¹⁰ mol min⁻¹ mg⁻¹) for SF–ZN–GSNO fibers was observed at 4 h, whereafter the NO release rate was declined over time and finally reached 0.09 ± 0.01 × 10⁻¹⁰ mol min⁻¹ mg⁻¹ at 48 h. The SF–ZN–GSNO fibers released their NO content in a more controlled manner and demonstrated significantly higher release rates compared to SF–GSNO fibers after 12 h (p < 0.05) and 24 h in media (p < 0.01). This is most likely caused by the enhanced hydrophobic properties of SF–ZN–GSNO fibers, leading to reduced water uptake and consequently slower release of NO in the media.

Figure 8.

Real-time NO release profile of SF–GSNO and SF–ZN–GSNO fibers in PBS with EDTA at 37 °C in the dark.

Excessive leaching of the NO donor molecule can adversely affect the longevity and cytocompatibility of the NO-releasing biomaterials. The cumulative amount of GSNO leached from the GSNO-loaded fibers into the media was measured at different time points through UV–vis spectroscopy, and the results are depicted in Figure 9. SF–GSNO fibers leached out 41.0 ± 1.5% of their GSNO content after 24 h, while SF–ZN–GSNO fibers had a significantly reduced GSNO leaching of 26.5 ± 2.2% (p < 0.001). This is associated with the presence of hydrophobic ZN in the structure that limits the diffusion of GSNO molecules into the aqueous media. According to these results, the SF–ZN–GSNO fibers offer a promising NO-releasing platform for biomedical applications as they minimize the leaching of GSNO from SF fibers and provide a sustained release of NO for over 48 h.

Figure 9.

Cumulative leaching of GSNO from SF–GSNO and SF–ZN–GSNO fibers over the course of 24 h when submerged in PBS at 37 °C.

3.8. Antibacterial Activity of the Fibers.

Bacterial infection is one of the main reasons for the failure of implantable biomaterials and may lead to serious consequences such as tissue damage, systemic infections, and patient morbidity and mortality.^51,52 Bacteria can adhere to the surface of medical devices upon implantation and encase themselves in EPS to form three-dimensional multi-cellular communities called biofilms. Bacteria in the biofilm are extremely difficult to treat as they are more resistant to host immune defenses and antibiotic treatments compared to their free-living counterparts.⁵³ According to previous reports, biofilms can form as quickly as 10 h, underscoring the importance of having an efficient antibacterial treatment in the first few hours of implantation.^12,54 NO-releasing biomaterials have shown effective antibacterial activity against different bacterial strains in the past.^29,55 NO targets the bacteria through various mechanisms including inhibition of protein synthesis, alteration of DNA, and damaging the membrane. The small size, short half-life, and non-specific multi-mechanistic bactericidal activity of NO make it an ideal antibacterial agent to combat antibiotic-resistant bacteria.⁵⁶ NO is also known to be able to disperse the bacteria in biofilms into a more vulnerable planktonic state by elevating the bacterial phosphodiesterase activity and therefore accelerating the degradation of c-di-GMP messenger molecules in charge of regulating the biofilm integrity.⁵⁷

In this study, an in vitro bacterial adhesion assay was performed to evaluate the antibacterial activity of the fibers against S. aureus and E. coli, two bacterial strains commonly found in nosocomial infections. The results are displayed in Figure 10. While pure SF fibers showed no antibacterial activity, SF–ZN fibers demonstrated a slight reduction in the viability of adhered S. aureus (32.5 ± 9.1%) and E. coli (24.2 ± 3.6%). The presence of abundant carotenoids such as lutein and zeaxanthin in ZN has been reported to endow it with antimicrobial activity to some extent.⁵⁸ While the exact antibacterial mechanism of action of these carotenoids has not yet been completely understood, they are thought to act on the bacterial membrane through increasing the rigidity of lipidic bilayers. In addition, lutein has been recently shown to be able to interact with LasI, LasR, RhlI, and RhlR proteins involved in the quorum sensing process and interrupt biofilm formation.⁵⁹ Furthermore, the NO-releasing SF–GSNO fibers reduced the viability of S. aureus and E. coli by 69.9 ± 9.9% (p < 0.01) and 68.6 ± 4.7% (p < 0.001), respectively. Addition of both ZN and GSNO to SF fibers led to synergistically higher bacteria reduction efficiencies of 91.6 ± 2.5% against S. aureus (p < 0.001) and 77.5 ± 3.1% against E. coli (p < 0.05). These results indicate that SF–ZN–GSNO employs both active and passive strategies to inhibit bacteria, thus offering promising potential to be used as antibacterial scaffolds for prevention of infections.

Figure 10.

Antibacterial activity of fibrous scaffolds against viable adhered (A) S. aureus and (B) E. coli, measured by a cfu counting method after 24 h exposure.

3.9. Cytocompatibility of the Fibers and Morphology of Adhered Cells.

Despite its strong antibacterial activity, NO at high levels has been shown to be cytotoxic toward mammalian cells due to excessive nitrosative and oxidative stress that it can impose on cells. GSNO is an endogenously produced NO donor molecule in the human body that has previously shown excellent cytocompatibility with mammalian cells when incorporated in polymeric fibers.^12,13 To evaluate any possible cytotoxicity of the GSNO blended fibers, 3T3 fibroblast cells were exposed to the 24 h leachates obtained by soaking the fibers in media. As seen in Figure 11, no cytotoxic response was observed for any of the samples with or without GSNO. All samples maintained cell viability at a similar level or a higher level than controls (cells exposed to no fiber) (p > 0.05). SF and ZN are natural polymers with high biocompatibility that have been extensively studied in the past for tissue engineering and biomedical applications. As shown in this study, the leaching of GSNO did not affect the cytocompatibility of these fibers, demonstrating the safe application of SF–ZN–GSNO fibers as a biomaterial.

Figure 11.

Viability of 3T3 fibroblast cells after 24 h exposure to the leachates of fibrous scaffolds (p > 0.05).

A qualitative fluorescence staining assay was performed to further assess the cytocompatibility of the fibers in direct contact with cells. Fibroblast cells play a critical role in wound healing by producing collagen, creating a new matrix, and regulating wound contraction.⁶⁰ Therefore, their proliferation and successful attachment to tissue engineering scaffolds are of great importance for proper healing and tissue regeneration. The delivery of NO through polymeric platforms has been reported to have an enhancing effect on fibroblast proliferation, growth, and collagen deposition. In this study, the morphology of adhered cells to the NO-releasing fibers was studied after 48 h exposure (Figure 12). Fibroblast cells successfully attached to all fibrous mats and displayed intact nuclei and nanofibrous actin cytoskeletons as stained by blue DAPI and green phalloidin reagents, respectively. A higher density of cells with more stretched and expanded morphologies and pronounced actin filaments were observed on GSNO blended samples, implying that the NO release from the fibers provides a favorable environment for cells to attach and grow. These results are consistent with previous studies emphasizing the important roles of NO in modulating and improving the wound healing process.^61,62 Therefore, the NO-releasing SF–ZN–GSNO fibers can be used as a non-toxic and cytocompatible substrate for promoting wound healing and tissue repair.

Figure 12.

Fluorescence microscopy images of 3T3 fibroblast cells seeded on (A) SF, (B) SF–GSNO, (C) SF–ZN, and (D) SF–ZN–GSNO fibrous scaffolds after 48 h.

4. CONCLUSIONS

In this work, two naturally derived proteins, SF and ZN, were combined and electrospun with the NO donor molecule, GSNO, to fabricate novel antibacterial, biodegradable, and NO-releasing fibers for biomedical applications. SF and ZN showed great miscibility and intermolecular bonding, which was confirmed by DSC and FTIR analyses. The incorporation of ZN into SF fibers reduced the diameter of the fibers and enhanced their enzymatic degradation, making them more suitable for scaffolding applications. The tensile testing showed an increase in the EB and UTS of SF fibers with the addition of ZN and GSNO, respectively. Moreover, blending of ZN into SF increased the hydrophobicity of the fibers by introducing more non-polar amino acids into the structure, thereby decreasing the GSNO leaching amount. The increased hydrophobicity of SF–ZN–GSNO compared to SF–GSNO fibers also led to a more controlled NO release profile with mitigated burst release and a more prolonged duration of 48 h. The continuous release of NO along with the inherent antibacterial properties of ZN endowed SF–ZN–GSNO fibers with synergistic active and passive antibacterial activity, resulting in 91.6 ± 2.5 and 77.5 ± 3.1% reduction efficiency values against S. aureus and E. coli, respectively. The 3T3 fibroblast cell viability and adhesion assays also demonstrated that SF–ZN–GSNO provided a non-cytotoxic ECM-like substrate for cells to adhere to and grow on. Overall, SF–ZN–GSNO fibers demonstrated good cytocompatibility, an enhanced biodegradation rate, controlled release kinetics, great mechanical properties, and high antibacterial activity, which make them ideal to be used for tissue engineering and wound healing applications.