Fabrication of bioactive glass-introduced nanofibrous membranes with multifunctions for potential wound dressing

Abstract

Currently, a variety of polymer-based membranes are available, which differ in compositions and microstructures, but are far away from being used in the treatment of chronic, nonhealing wounds. Herein, we design new bioactive glass (BG)-introduced multifunctional gelatin/chitosan (G/C) nanofibrous membranes for chronic wound healing, due to the efficacy of the antibacterial and wound healing properties of chitosan and BG. The water contact angle of the mats increased and the water uptake capacity decreased with an increasing BG content, suggesting that their surface hydrophilicity can be adjusted by the BG component. The biologically active ions were readily released from the mats, which is potentially favorable for infectious wounds. Also, the G/C–BG mats were well tolerated by the surrounding host tissue without causing any inflammation and fully degraded by the subcutaneous tissue of rats after 4 weeks post operation. Therefore, the G/C–BG mats afforded a close biomimicry to the fibrous nanostructure of natural soft tissues to facilitate chronic, nonhealing wound treatment.

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1. Introduction

In wound operations, the purpose of dressing the wound is to promote an optimal healing environment by providing pain relief, protection from trauma and infection, a moist environment, and removal of debris. By simultaneously maximizing the patient’s nutritional status and providing meticulous wound care, most wounds will heal appropriately.¹ However, the chronic, nonhealing wounds are involved progressively in tissue loss and bacterial colonization, particularly in venous stasis, diabetic ulcers, and bed sores, and thus give rise to a big challenge to wound-care product researchers.^2–4

It is known that calcium is an important factor in the wound healing of skin and it is assumed to be required for the migration of epidermal cells.⁵ Clinically, the direct topical application of calcium to chronic wounds through calcium alginate dressings has been shown to be beneficial.⁶ On the other hand, bacterial invasion may make the wound unsuitable for skin regeneration due to the growth of a bacterial population. So, the goal of wound dressing is to produce an ideal structure with high porosity and antimicrobial activity, which can also be a good barrier. Recently, some studies have found that the local release of inorganic ions from bioactive glasses (BGs) can suppress microorganisms, stimulate collagen production, and accelerate the wound healing process.^7–10 It was found that BGs can be combined with soft tissue and promote soft tissue regeneration.¹¹ Moreover, BG can also reduce the inflammatory response and wound exudate, and has a certain antibacterial and hemostatic potential, which has a positive therapeutic effect on the wound.¹² Wilson et al. firstly showed that soft connective tissues could form a bond to 45S5 Bioglass® and established the safe use of particulate forms in soft tissue if the interface was immobile.¹³ More recent studies demonstrated the beneficial effects of 45S5 Bioglass® and borate-containing 13–93 BGs to promote angiogenesis, which is critical to the healing of soft tissue wounds.^14,15 Thus, the development of BG-loaded porous wound dressings with antimicrobial activity is highly desired.

Electrospinning is a versatile technique for bridging the gap between the natural extracellular matrix (ECM) and artificial materials. With its simple manufacturing equipment, low cost, and controllable process, electrospinning has become one of the main approaches to prepare nanofibrous materials. It offers a range of benefits, including the ability to produce continuous nanofibers and controllable ECM-mimicking nanostructures, and thus enhances the cell migration and proliferation.¹⁶ Wound dressings via electrospinning can meet the requirements, such as higher gas permeation and protection of the wound from infection and dehydration, for electrospinning materials and have a higher porosity and huge surface area.¹⁷ It is also important to select the appropriate materials to guarantee the biocompatibility and biodegradability of the wound dressings. Gelatin has an excellent biocompatibility and cell adhesion ability, and is widely used in clinics for wound dressings, pharmaceuticals and adhesives due to its biodegradability and hydrogel characteristics, as well as its formability and cost efficiency.¹⁸ However, gelatin, as a kind of hydrolysis derivative of collagen, possesses quite a number of ionizable groups, and additionally, strong hydrogen bonding results in a three-dimensional (3D) macro-molecular network, which makes the mobility of the gelatin chains decrease tremendously and gives it poor electrospinnability.^19,20 Chitosan is a naturally positively charged basic polysaccharide and has been shown to exhibit some pharmacological activities, including antimicrobial, anti-inflammatory, anti-adhesion, and wound healing effects.²¹ Javad et al. has prepared gelatin/chitosan (G/C) nanofibers using an acetic acid solution.²² However, it is still a great challenge to treat chronic, nonhealing wounds with these pure polymeric materials.^2,23

Most recently, we developed a nanofibrous gelatin (NF-GEL)/BG composite hydrogel using a phase separation method, which was followed by arming the nanofiber network with counterionic chitosan–hyaluronic acid pairs for improving the microstructural and thermal integrity.²⁴ This NF-GEL-based hydrogel could afford a close biomimicry to the fibrous nanostructure of the natural soft tissues, but its very high water uptake capacity (∼900%) is potentially disadvantageous for the accumulation of fluid in the moist wound areas. In this regard, herein we designed a new G/C-based nanofibrous membrane as a wound dressing with high biocompatible and bioactive properties (see Scheme 1). We explored the fabrication of G/C membranes in the presence of BG superfine particles. The BG with a composition of SiO₂–CaO–B₂O₃–P₂O₅–CuO–ZnO–K₂O–Na₂O was chosen based on its specific biological performances in the wound healing process. Calcium, potassium, and sodium are well known to be essential for promoting epithelial repair during the wound healing process. Moreover, both copper and zinc are suggested to have the ability of promoting skin regeneration and to have an antimicrobial activity in the wound healing process.^25,26 Boron may modulate the turnover of the extracellular matrix and enhance the collagenase and cathepsin D activities in fibroblasts.²⁷ To our knowledge, this study is the first to investigate the electrospinnability of a multi-component BG-introduced G/C aqueous solution for wound dressings. Furthermore, its physicochemical properties and biocompatibility were studied for the potential treatment of chronic wound healing.

Scheme 1 Schematic illustration of the preparation procedure of the BG-introduced G/C nanofibrous membranes.

2. Materials and methods

2.1 Materials and chemicals

Chitosan (MW, 50 kDa) with a degree of deacetylation of 90% was purchased from Haidebei Marine Bioengineering Inc, China. Gelatin (type B, from bovine), tetraethyl orthosilicate (TEOS; 99.99%), triethyl phosphite (P(OEt)₃; 99.50%), and the inorganic salts were purchased from Sinopharm Chemical Reagent Co., China. All the chemicals were of analytical grade and were used directly without further purification.

2.2 Synthesis of BGs

The BGs with 30.0 SiO₂, 27.0 CaO, 20.0 B₂O₃, 4.0 P₂O₅, 1.5 CuO, 1.0 ZnO, 3.0 K₂O, and 9.0 Na₂O (wt%) were prepared by a sol–gel process according to our previous work.²⁸ In brief, HCl, H₃BO₃, TEOS, and P(OEt)₃ were added into an ethanol–water mixture and stirred for 20 min. The nitrates of calcium, sodium, copper, zinc, and potassium were then added into deionized water (DI water) under magnetic stirring for 20 min. The solutions were mixed and subsequently, aged at 80 °C for 36 h and calcined at 680 °C for 90 min. The as-obtained BG powders were ball milled for 4 h to reach a smaller particle size and characterized by scanning electron microscopy (SEM; JEM-6700F). The particle size distribution data were collected by DLS in a Mastersizer 2000 analyzer (Malvern Instruments Ltd.).

2.3 Electrospinning of G/C–xBG membranes

Gelatin was dissolved in DI water to form a concentration of 25 wt% and chitosan was dissolved in an acetic acid solution (70 wt%) to form a concentration of 3.0 wt%. Then, the gelatin solution, chitosan solution and BG powders were mixed with different ratios under magnetic stirring to form an organic–inorganic hybrid suspension (Table 1).

Table 1 Composition of the electrospinning solutions

Samples	Gelatin (g mL^−1)	Chitosan (g mL^−1)	C/G (m m^−1)	BG/(G + C) (m%)
G/C19	17.23	0.91	1/19	0
G/C17	9.94	1.75	3/17	0
G/C15	6.64	2.16	5/15	0
G/C–0BG	9.94	1.75	3/17	0
G/C–6BG	9.94	1.75	3/17	6
G/C–12BG	9.94	1.75	3/17	12
C/C–15BG	9.94	1.75	3/17	15

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The suspension solutions with a chitosan/gelatin (C/G) ratio of 1 : 19, 3 : 17 and 5 : 15 in the presence and absence of BGs were poured into a 15 mL plastic syringe equipped with a 20-gauge metal needle, which was then placed on the infusion syringe pump to modulate the pump rate at 0.2 mL h⁻¹. The needle was connected to a high-voltage generator (0–50 kV; Dongwen High Voltage Inc., China). The device was placed in a chamber that provided flowing warm air. A 13 kV voltage was used, and the grounded Al collector was placed at 12 cm from the needle tip. The fibers were electrospun onto the collector and formed the G/C–xBG [x = 0, 6, 12, 15%; x is denoted as the BG/(G + C) percent ratio in the electrospinning solutions] fibrous membranes.

2.4 Chemical cross-linking of the G/C–xBG membranes

After the electrospun membrane was dried in vacuum for 24 h, glutaraldehyde (GA) vapour cross-linking was carried out by placing the membrane above the GA solution (25 wt%) in a sealed desiccator at room temperature for 48 h.²⁹ Then the GA-cross-linked G/C–xBG membranes were washed three times with distilled water and dried in vacuum. The microstructures of the G/C–xBG membranes were characterized by SEM and energy dispersive spectrometry (EDS). The thermal analysis of the membrane samples was carried out with a heating rate of 10 °C min⁻¹ from 20 °C to 900 °C in air, using a thermogravimetric and differential thermalgravimetric analysis (TG-DTA; Perkin Elmer, USA). FTIR spectra of the samples were obtained using Fourier transform infrared spectroscopy (Nicolet). The spectra were collected over the wave number range of 400–4000 cm⁻¹.

2.5 Mechanical characterization

The mechanical properties of the samples were characterized using a multipurpose tensile tester (KES-GI; Kato-Tech, Japan) in tensile mode.³⁰ Firstly, a white paper was cut into a template with width × gauge length, and double-sided tapes were glued onto the top and bottom areas of one side. The mats with a planar dimension of 5 mm × 40 mm were then glued onto the top and bottom sides of the paper template along the vertical lines. Each sample was firstly stored under 65% relative humidity at 20 °C for 24 h and then tested at 2 mm min⁻¹ elongation speed for four times.

2.6 Water contact angle (WCA) measurement

The static WCA of the G/C–xBG membrane was measured by using a DSA100 drop shape analysis system (Krüss, Germany). An image was taken with the built-in digital camera. The average static WCA was obtained from three points of each sample and 3 μL deionized water was used in each experiment (23 °C).

2.7 Equilibrium water uptake capacity (WUC) and area change ratio (ACR) measurements

The swelling ratio was determined by immersing the porous membranes in MiniQ water at 37 °C for 4 h and was considered to reach the equilibrium of water uptake. The WUC and ACR were quantified as: WUC (wt%)=(W − W₀)/W₀ × 100 and ACR (S%) = S_s/S_d × 100, where W₀ and W are the dry weight and wet weight of the samples before and after immersing them in water and removing the surface water with filter paper, respectively; S_d and S_s are the original area of the membranes and the measured area of the swelled membranes after immersion in water, respectively.

2.8 Inorganic ion release test in vitro

The inorganic ion release was investigated for the G/C–xBG membranes (5 cm × 5 cm; ∼150 μm in thickness) by immersing them in 5 mL Tris buffer (0.02 M) with initial pH 7.20 at physiological temperature in vitro. After immersion for different time intervals, the changes in pH of the aqueous medium were measured firstly; then 0.5 mL of the supernatant was diluted in 5% HCl solution, and an aliquot amount of fresh buffer (0.5 mL) was added into the buffers to maintain a constant solution volume. The inorganic mineral elements, including calcium (Ca), zinc (Zn), copper (Cu), boron (B), and silicon (Si), in the supernatant were determined using an inductively coupled plasma spectrometry (ICP; IRIS INTREPID II XSP, Thermo) analysis.

2.9 In vitro antibacterial test

The direct antimicrobial activity of the BG disks (Ø 6 mm × 2 mm) was tested by agar diffusion using Actinomyces viscosus (A. viscosus) and Escherichia coli (E. coli). The disks were prepared in a stainless steel mould and sintered at 650 °C for 90 min. Sintered disks of commercially available 45S5 Bioglass® (US Biomaterials Co.) were used as a control. The disks were allowed to set at 37 °C and 100% humidity for 24 h. The bacterial strains were maintained as subcultures on trypticase soy agar (TSA) plates at 37 °C in an anaerobic chamber, where O₂ and H₂ were purged down to 100 ppm and 1%, respectively, using N₂ and a biological atmosphere mixture (5% CO₂ in N₂). The BG disks were placed at the centre of the agar plate and the inhibition zone around the disks was observed by SEM observation after incubation for 16 h.

2.10 In vivo biocompatibility test

The procedures for the use of animals were in accordance with the animal care and use committee of Zhejiang University. Prior to implantation, the membranes were UV light sterilized from different directions. Each side was sterilized for 45 min. Adult Sprague Dawley (SD) rats, weighing 250–380 g, were anaesthetized with 0.25 mL IM Hypnorm (0.315 mg mL⁻¹ fentanyl citrate and 10 mg mL⁻¹ fluanisone) and 1 mg IP diazepam. A midline incision of 2–3 cm was made through the shaved skin on their backs.³¹ The G/C–0BG and G/C–15BG membranes were then implanted into the subcutaneous pockets on the right and left hand side of the incision. After 1, 2 and 4 weeks of implantation, the rats were sacrificed using pentobarbitone (200 mg mL⁻¹), and optical images of the membranes and subcutaneous tissues were taken.

2.11 Statistical analysis

The results were expressed as mean ± standard deviation (mean ± SD). The statistical significance was tested using a one-way ANOVA test, and differences of p < 0.05 were considered to be statistically significant.

3. Results and discussion

3.1 Morphology characterization

It is known that a pure gelatin solution is usually in a hydrosol state at above physiological temperature, and when the gelatin concentration is high enough and its temperature is decreased to below 37 °C, the hydrosol transforms into a hydrogel. Thus, the introduction of another polymer component (e.g. chitosan) into the gelatin matrix is of high significance because the pure gelatin fibers are very brittle and unstable in an aqueous environment. Fig. 1 shows the SEM images of the electrospun G/C mats with a different C/G weight ratio (1/19, 3/17, and 5/15). It was evident that the G/C membranes were composed of open pores with several microns in size, and the pore wall was made of thin fibers. With increasing the C/G ratio from 1/19 to 5/15 in the solutions, the beads could be seen in the fiber body, and were prone to aggregation. It should be mentioned that the parameters, such as the electric field and the spinning distance, were examined for studying the effects of the C/G ratio in the solutions (i.e. 3/17, 5/15, and 6/14) on the electrospinnability and morphology of the gelatin at a concentration of 17.23 g mL⁻¹. It was found that gelatin nanofibres were hardly obtained until the gelatin concentration was reduced to 9.94 and 6.64 g mL⁻¹ and the weight ratio of chitosan and gelatin was increased to 3/17 and 5/15. Furthermore, when the weight ratio of chitosan and gelatin exceeded 6/14, it was almost not possible to get well shaped fibres (not shown). These primary results suggested that the electrospinning of the G/C composite fibers mainly works at a C/G ratio between 1/19 and 3/17. In order to endow the multifunctions of the composite membranes, the electrospun G/C-based fibrous membranes with a C/G ratio of 3/17 and an appropriate amount of BG addition is highly interesting in both fundamental and practical aspects.

Fig. 1 SEM images of the G/C nanofibrous mats with different C/G mass ratios.

The sol–gel method is versatile to prepare nanoscale BG powders in comparison to the high-temperature calcination treatment. The measured composition of the powders was 30.4 SiO₂, 27.2 CaO, 19.6 B₂O₃, 3.7 P₂O₅, 1.6 CuO, 1.1 ZnO, 3.2 K₂O, and 8.7 Na₂O (wt%), which was similar to the theoretical values mentioned above. The SEM observation in Fig. 2A shows that the sol–gel-derived BGs were highly dispersible with a nanoscale dimension particle size. Meanwhile, the quantitative analysis confirmed that the BG powders had a very narrow particle size distribution in 840–1660 nm (Fig. 2B), suggesting that BG is favorable for electrospinning with an organic component.

Fig. 2 SEM observation (A) and particle size distribution (B) of the BG powders.

According to the SEM observation (Fig. 3A–H), the G/C–xBG membranes with a differently introduced amount of BG at C/G ratio 3/17 via the electrospinning technique showed some differences in the fiber morphology with an increasing BG/(G + C) ratio from 6% to 15%, but the fiber diameter ranged from tens to hundreds of nanometers, which was the same range as that of natural collagen matrices. The mats grew some blade-like continuous fibrous networks with BG/(G + C) increasing up to 12–15%, and the BG particles were anchored in the fiber matrix and were scarcely exposed in the porous network with an increasing BG content. It is also noted from the EDX analysis (Fig. 3K–N) that Si could be detected in the BG-introduced membranes, and the intensity of the silicon peak increases with the increase of BG content in the membranes. It should be mentioned that the BGs with appropriate inorganic oxide compositions are highly beneficial for antibacterial activity and soft tissue regeneration in chronic wounds.^7–10 Unfortunately, the mixture solution is poorly electrospun with an increasing BG/(G + C) ratio of up to 18%. Accordingly, these results suggest that the appropriate amount of BGs (i.e. 15%) could be successfully nested in the G/C nanofiber body.

Fig. 3 SEM observation (A–H) and EDX analysis (K–N) of the G/C–xBG membranes with introducing a different amount of BG at C/G ratio 3/17 via the electrospinning technique.

3.2 FTIR analysis

The G/C–xBG membranes and BG powders were characterized by FTIR to investigate the molecular changes after adding BG into the G/C nanofibrous mats. As illustrated in Fig. 4A, the FTIR spectra of all membrane samples had a similar roughness. Amide I (∼1650 cm⁻¹) and amide II (∼1550 cm⁻¹), which are common protein bands and correspond to the stretching vibrations of the C=O bond and coupling of the bending of the N–H bond and stretching of the C–N bonds,³² were attributable to gelatin which is derived from collagen by controllable hydrolysis. Other distinct bands, such as C=C (∼1410 cm⁻¹) and C–O (∼1038, 1241, and 1080 cm⁻¹) were contributed to gelatin and chitosan. The FTIR spectra of BG reveal bands at the Si–O–Si bend and the P–O stretch at 462 cm⁻¹ and 1037 cm⁻¹, respectively.³³ In Fig. 4B, the FTIR spectra of BG, G/C–0BG and G/C–15BG were put together to show the difference. In particular, in Fig. 4C, which is a magnified spectrum from 400–550 cm⁻¹, the Si–O–Si bend peak is shown from the spectra of BG and G/C–15BG, but not of G/C–0BG, which is made up of pure polymers. These results suggest the the superfine BG particles are readily introduced into the electrospun G/C composite fibers.

Fig. 4 FTIR spectra (A) of the G/C–xBG nanofibrous mats. The magnified FTIR spectra (B and C).

3.3 TG-DTA analysis

The G/C–xBG mats and BG powders were further analyzed by TG-DTA (Fig. 5). From the DTA curve of BG, no strong exothermic or endothermic behavior was seen. Furthermore, the total weight loss of BG was about 22%, mainly due to the removal of the adsorbed moisture and the pyrolysis or decomposition of residual nitrate groups and organic components during the sol–gel synthesis process.³⁴ As for the G/C–xBG membranes, there was an endothermic event at 35–150 °C with a weight decrease of 13%, due to the loss of adsorbed water. The significant weight loss (up to 76–83%) is recorded in the temperature range of 200–530 °C, and this is accompanied by a strong exothermic peak at 450–530 °C, which is caused by the pyrolysis of the gelatin and chitosan components. Evidently, the mass residual of the G/C–6BG, G/C–12BG and G/C–15BG mats were consistent with the introduced mass percent of the BG in the precursor solutions. By increasing the amount of BG in the G/C mixture solutions, the strong exothermic peak has a tendency to be at a lower temperature and the remains tend to increase after the entire thermal weight loss process, compared to G/C–0BG.

Fig. 5 TG (A) and DTA (B) curves of the G/C–xBG nanofibrous mats and pure BG.

3.4 Mechanical analysis

The tensile strength was measured to confirm the operability of the mats. Fig. 6 shows the representative stress–strain curves, and Table 2 shows the mechanical data and elongation at break. It can be seen that the BG-introduced mats exhibited a considerable tensile strength (up to two to four fold higher than that of the pure organic mats), meanwhile the increase of the BG content produced a further increase in mechanical strength, indicating that the inorganic–organic interaction between G/C and BG could reinforce the mechanical stability of the mats. More precisely, the average elongation ratio of the mats increased by nearly 150% after the addition of 15% BG, thus making it considerably favourable for biomedical applications.

Fig. 6 Representative stress–strain curves of the G/C–xBG nanofibrous mats (the insets show the optical images of the mats before and after the mechanical test).

Table 2 Mechanical data of the electrospun mats

Samples	Tensile strength (MPa)	Young’s modulus (MPa)	Elongation at break (%)
G/C–0BG	2.05	132	1.41
G/C–6BG	4.00	268	1.39
G/C–12BG	5.00	244	2.39
C/C–15BG	7.92	269	3.56

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3.5 WCA analysis

It is well agreed that hydrophilicity influences the adsorption of blood proteins, and especially the cell attachment behavior may be regulated through these proteins.³⁵ Samples which produce small contact angles with blood are, in general, associated with good blood compatibility, whereas those with large contact angles have a relatively high anti-adhesion ability.³⁶ Thus, the surface-responsive behavior of the electrospun mats with different BG contents was analyzed by measuring the static WCA (Fig. 7). The pure organic G/C–0BG mats were quite hydrophilic, presenting an average contact angle of 20.4°. In contrast, the hydrophobicity of the organic–inorganic hybrid mats was substantially created by the BG/(G + C) percent ratio in the mats. The contact angle was slightly increased to 47.4° after introducing 6% BG, and significantly increased to 84.9° and 94.6° when adding 12% and 15% BG, respectively, which was related to the role of the microscopic BG particles embedded in the nanofibers. This is consistent with the fact that the WCA measurements are significantly affected by several parameters, namely the topographical roughness and composition. By looking at the SEM images in Fig. 3, it is evident that the roughnesses of the G/C–xBG mats without and with BG are different, at least at the submicron length scale. In fact, the hydrophilic to hydrophobic transition phenomenon for the hybrid mats is attributed to relatively rougher surface, and in particular the responsive wettability change is greatly amplified on rough substrates with micro- and nano-structures, and increases significantly with the increase in roughness.^37,38 This means that the hydrophilicity of the electrospun mats can be adjusted by changing the amount of the BGs.

Fig. 7 Water contact angles (WCA) of the G/C–xBG nanofibrous mats (*p < 0.05 significantly different from G/C–0BG; **p < 0.01 significantly different from G/C–0BG).

3.6 WUC and ACR analysis

It is known that the wound areas should be kept just moist enough to obtain the benefits of accelerated healing, but there should be no accumulation of fluid between the wound and the dressing to avoid infection. Fig. 8 shows the WUC and ACR, which had opposite trends with an increasing BG content. The polymer membrane that was free of BG after the cross-linking treatment had the highest WUC value (721.15 ± 21.30%), and its ACR was 98.28 ± 0.75 v/v%. By contrast, the WUC of the sample with the highest BG/(G + C) ratio (15%) was reduced to 217.8 ± 10.8%, but its ACR changed only a little (99.64 ± 0.47%). These results suggest that the cross-linked G/C–xBG mats become more rigid and the incorporation of BG in the polymer matrix readily reduces its water uptake capacity.

Fig. 8 Water uptake capacity and area change ratio of the G/C–xBG nanofibrous mats (**p < 0.01 significantly different from G/C–0BG).

3.7 Inorganic ion release analysis

To distinguish the contribution of the BGs for the inorganic ion release, different immersion times for the G/C–xBG mats with different BG contents were investigated (Fig. 9A–E). As for the G/C–15BG sample, the rapid calcium and boron accumulation took place in the aqueous media within the initial 4 h, accompanied by a steady release of silicon, zinc and copper. Also, the copper and zinc concentrations were over 2.0 ppm and 0.8 ppm after immersion for 24 h, indicating that the antibacterial inorganic ion dose in the immersion media can be accelerated with prolonging the immersion time. In contrast, the inorganic ions were hardly measured in the medium during soaking the G/C–0BG mat. Indeed, the overall concentrations of the calcium ions for G/C–15BG increased markedly from the initial zero value to nearly a two-fold of calcium ions for the G/C–6BG sample in the media within 8 h, and thereafter both kept stable without much fluctuation (not shown).

Fig. 9 Ion release from the PBS medium (A–E) and changes in pH (F) when immersing the G/C–0BG and G/C–15BG mats for different times.

It is seen that the pH value in the medium, in which G/C–15BG was immersed, showed a rapid increase from the initial value of 7.2 to 8.6 within 8 h (Fig. 9F). The greatest rate of pH increase was recorded within the first 4 h, and within 24 h a plateau (pH ∼ 9.0) was reached, which would be effective in inhibiting the growth of some bacteria.^8,9 However, the pH value in the Tris buffer, in which the pure polymer mats were immersed, showed a slight decrease (∼7.05) within the initial two hours and then slightly increased to nearly 7.2. This is possibly attributed to the acetic acid residual, which has been used to dissolve chitosan. Moreover, it is stressed that the ion concentration values varied depending on the BG content and similar trends in pH change were observed for all of the G/C–xCu mats (not shown).

3.8 In vitro antibacterial evaluation

The BG formulations inhibited growth of A. viscosus and E. coli, as clear rings appeared around the disk samples inserted into the agar plate. The BG markedly inhibited the growth of A. viscosus, producing a larger zone of inhibition than the 45S5 Bioglass® (Fig. 10A). Meanwhile, the 45S5 Bioglass® appeared to cause a much lower inhibition effect on E. coli than the BG containing CuO and ZnO; for instance, the 45S5 Bioglass® had no substantial effect on the bacterial viability of E. coli (Fig. 10B), similar to the results reported previously.³⁹ These results also demonstrate that an appropriate level of ZnO and CuO introduction in BG have a considerable effect on inhibiting the microbial viability.

Fig. 10 Agar diffusion test of the BG and 45S5 Bioglass® formulation with A. viscosus (A) and E. coli (B) bacterial strains.

3.9 In vivo biocompatibility evaluation

Gelatin is available via a controlled hydrolysis of collagens, which is one of the most abundant structural fibrous insoluble proteins throughout the body. The gelation temperature of GEL is very close to the human physiological temperature, and accordingly the gelatin-based dressings must be cross-linked to improve its thermal and structural stabilities in a wet state. As shown in Fig. 11, both of the GA-cross-linked G/C–0BG and G/C–15BG mats were well tolerated by the surrounding host tissue without causing any inflammation in the rat subcutaneous tissue after one and two weeks of implantation, which indicates that the mats are highly biocompatible. Interestingly, the G/C–0BG and G/C–15BG mats were degraded in the subcutaneous tissue after 4 weeks of implantation, implying that the mats are of good biodegradability. It is reasonable to postulate that the G/C–6BG and G/C–12BG mats show similar performances. The characterization of the biocompatibility and biodegradability of the nanofibrous mats shows a perfect match for the needs of wound dressing. Evidently, the excellent biocompatibility of the mats is due to the intrinsic quality of the ingredients and the nanofibrous structure.

Fig. 11 Optical pictures of the implanted G/C–xBG mats in the subcutaneous tissue of the rat model.

It is known that biomimetic processes and especially high voltage electrospinning is well suited to generate nanofibrous porous membrane matrices directly from polymer solutions. Until now, many researchers have examined gelatin, chitosan, and BG as promising materials for the acceleration of wound healing. Unfortunately, gelatin exhibits poor mechanical stability that is too brittle when fully dried or too soft when fully wet and is easily soluble in aqueous medium. On the other hand, chitosan is obtained by the hydrolysis of chitin and is the only cationic polysaccharide in nature, with an outstanding wound healing effect.⁴⁰ Therefore, the primary objective of this study was to develop a G/C-based electrospun membrane dressing with improved biologically active and antibacterial properties. The ionic form of silver (Ag⁺) is a well-known highly antibacterial material, and the silver-loaded dressing is also an increasingly popular approach in the control of wound bioburden.⁴¹ However, a high concentration of silver impairs the functioning of the central and peripheral nervous systems.^42,43 In contrast, a variety of studies have demonstrated the angiogenic effects of BG, i.e., increased secretion of vascular endothelial growth factors (VEGFs) and VEGF gene expression in fibroblasts, the proliferation of endothelial cells and formation of endothelial tubules in vitro, as well as the enhancement of vascularisation and wound healing in vivo.^44,45 Furthermore, BGs may inhibit the growth of a wide selection of aerobic and anaerobic bacterial species, typically causing infections on the surface of prostheses, and the antibacterial activity of BG correlated mainly with the ion doping, particle size, pH and high silicon ion levels in the supernatant.^9,46 Moreover, the ion product (e.g., Ca²⁺, Na⁺, and K⁺) dissolution from BGs have been demonstrated to be favorable for improving the electrospinnability of chitosan in our previous studies.⁴⁷ Therefore, the introduction of BG into the G/C composite membranes is highly desirable and beneficial for improving the chronic, nonhealing wound treatment.

It is assumed that the bioactive potential of the G/C–xBG membranes can be attributed to two aspects: the inorganic ion products from the BG dissolution and the beneficial functions of G/C on the wound surface. These two constituents may be advantageous to mediate their activity in cellular signaling. Meanwhile, the BGs in the nanofibrous network provide a high antibacterial activity and modified surface properties, which would be helpful for anti-adhesion in wet wound areas. Therefore, it is postulated the introduction of BG endows improved biological performances for chronic wound healing. These results further support the hypothesis that the nanofibrous membranes have the potential ability to enhance wound healing in pathological animal models. These studies will be presented in the near future.

4. Conclusion

In summary, G/C–xBG nanofibrous composite membranes were successfully prepared using chitosan and gelatin dissolved with acetic acid and water, respectively. Then BG particles were nested in the nanofibers, which readily adjusted the hydrophilicity of the electrospun mats. The inorganic ion release from the BG dissolution endows the materials with bioactive and antibacterial properties. The whole manufacturing process does not involve environmentally/biologically harmful additives and the mat products exhibit an excellent biocompatibility, without involving any inert constituents. Therefore, this composition–structure relationship makes G/C–xBG nanofibrous mats more attractive candidates for potential uses as wound-healing accelerators in many chronic, nonhealing skin wound areas.

Acknowledgements

This work is supported by the National Science Foundation of China (51102211, 81301326, and 81271956), the Zhejiang Provincial Natural Science Foundation of China (LZ14E020001 and LQ14H060003), the Fundamental Research Funds for the Central University (2012QN81001), Research Fund of Zhejiang Provincial Education Department (Y201016210), and Shaoxing Science and Technology Bureau Foundation (2012B70016).

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