A novel fabrication of PVA/Alginate-Bioglass electrospun for biomedical engineering application

INTRODUCTION

There has been an interesting attention in biodegradable and nonbiodegradable polymers materials proposed for nanomedicine applications [1-2]. To produce excellent soft/hard scaffolds, the important properties are proper biological, mechanical, chemical stability, great porosity, and a condition that allow the cells organisms to grow and proliferate on the surface of the tissue [3]. It is well recognized that micropores fibers have suitable interconnectivity and can play significant rule in the growth cellular on the bone and help the regeneration of tissues [4]; however, improving the fibers porosity is occurring with decreasing the mechanical properties. Therefore, various types of polymers like poly vinylalcohol (PVA) [5, 23], poly acrylamide (PAC) [6], and poly acrylicacid (PAA) [7] have been evaluated for soft/hard artificial tissue application by several researchers. Among the investigated polymers, PVA has been implemented for nanomedicine applications better than other polymers [8-12]. The PVA has interesting properties like suitable nontoxicity, hydrophilicity, food safe degradation, mechanical properties, biocompatibility, high degradation in water, and bioadhesive characteristic. The PVA can applied for regenerative medicine especially wound skin healing, optical, chemical destruction and degradation products which are excreted without any harm to the body [13-15].  Another known polymer, alginate is used to mix with nanomaterials to enhance the behaviour of based polymers. In addition, the alginate has been used as an emulsifier, pharmaceutical products and wound healing applications [15]. The alginate and PVA represent renewable sources and do not have adverse impact on skin and eye irritation. It is known that alginate have proper ductility, commercial availability and high efficiency that will facilitate manufacturing capabilities [16-17]. In this study, the nanocomposite PVA/alginate/bioaglass (PVA/Alg/BG) is reconstructed. Notwithstanding the weak mechanical properties of polymer materials, the excellent biology behavior of BG and alginate attracts our attention to focus on enhancing the properties of the PVA polymer for biomedical application. The BG bioceramics has recently been taken into consideration due to its good chemical and mechanical stability. Adding BG to biopolymer-based nanocomposites improves their mechanical, biological, chemical, thermal properties of polymers [18-21]. The electrospinning (ELS) technique common used method of making polymer scaffolds for tissue engineering approaches. ELS technique is simple, economic and commercializes method in biomaterials field for preparation of soft tissues [22-24]. The polymer fibers prepared by ELS technique have excellent mechanical properties, flexibility and biocompatibility inside the human’s body.  Therefore, we have prepared a novel PVA/Alg/BG nanocomposite for the first time as a bioactive material for wound healing applications.

 MATERIALS AND METHODS

Starting materials poly vinylalcohol (PVA) (M_w=145,000) (Merck Co, Germany), Alginate (Aldrich Co., USA) and bioglass (BG) (Nikceram, Iran Co.) were purchase. The chemistry analysis of bioglass were assessed with X-ray fluorescence (XRF) test and represented in Table 1.

Composite Preparation

Four polymer scaffold nanocomposite solution were prepared with different ratios 100, 90:10, 85:10, and 80:10 of PVA/Alginate in 100 ml of deionized water. The BG particles with 5% and 10% (w/w) were added to the nanocomposite solutions. The influence of BG on the biopolymers was investigated by FTIR and SEM analysis that equipped by EDX software. The BG powder was then slowly added to the PVA/Alginate solution on stirrer at room temperature (T=37°C). The solution was allowed to mix for 1 h in a sealed container and then obtained mixture was further sonicated for 20 min.

Electrospinning Technique

The prepared scaffold nanocomposite solution were separately added by dispersion of PVA and Alginate in distilled water and then stirred. Then, ELS technique as one of the most important approaches for developing continuous nanoscale fibres with diameters ranging from 10-50 nm were used to the sample. Each sample solutions were mixed with PVA solution at constant volume ratio. The prepared solutions were applied into a 5 mL syringe of ELS tools as represented in schematic Fig 1. The condition for ELS were selected at room temperature (T=37°C) according to the procedure described by Heydari et al. [1, 8]. The feed rate setting for ELS adjusted at 0.25 mL/h. Then the nanofiber nanocomposite scaffolds produced on aluminum foil rotated drum. The collector distance from tip of needle set at 15-20 cm. The adequate high voltage was choosing at 15 with step of 5 kV. The optimum voltage observed to select was 25 kV. Also, the dimension of frame was 10×50 mm² which was positioned at a constant distance of 10 cm from the needle. Finally, the prepared scaffolds dried at 50-60°C in the vacuum condition for removing the moisture and the residual solvent of scaffolds. The ELS procedure (compositing (PVA, Alginate, and BG), syringe pump, and electrospun on aluminum foil) is shown in Fig. 1.

Biological Testing

Bioactivity Test

To recognize the biological response of scaffold nanocomposite the specimen are soaked in the simulated body fluids (SBF). According to the Kokubo's test, the important requirement for any materials to mimic and bond with the host tissue can be recognized by the formation of apatite (bone-like/cauliflower) on its surface after the implantation to the live part [25-28]. The formation of apatite on the surface of scaffold nanocomposite of polymer/bioglass show proper ability to precipitate apatite (cauliflower) on the surface of samples [28-30]. Table 2 shows the SBF ion concentration solution according to the Kokubo's test and the comparison of SBF with human blood plasma. The SBF were fabricated according to the procedure described by Kokubo et al. [31]. In this study, the nanocomposite scaffold samples were immersed in the SBF solution for the certain period times of 1, 2, and 3 weeks at 37°C with pH = 7.4 to investigate bioactivity and biodegradation of samples.  The samples were dried for 45 min at 50°C after the soaking in the SBF and weighted to record the weight changes (weight loss).

The degree of the weight loss of the nanofibre samples was calculated by the use of Eq. (1). The test is carried out in the phosphate buffered saline, PBS, (pH=7.4) at 37°C for 1, 2, and 3 weeks. The PBS solution was prepared by dissolving the NaCl (8 g), KCl (0.2 g), Na₂HPO₄ (1.44 g), and KH₂PO₄ (0.24 g) in 800 ml deionized water according to the Table 2. The pH of PBS prepared at room temperature has to be adjusted at 7.4. The nanocomposite scaffolds were inserted in a 20 ml falcon tube with 15 ml PBS solution. The produced samples were then placed in sonicate water bath at 37 ±1 °C for 3 weeks. Each week, the solution pH was measured by pH metre. Weight change was calculated by this formula

˟100           Equation (1)

W₀ = initial dry weight sample and W_t = dry weight sample after specific time.

 Mechanical Properties

The PVA/Alginate nanocomposite scaffold with 5% and 10% BG nanoparticles were tested for mechanical testing. The mechanical properties of fibres including tensile strength and elongation at fracture point are measured by tensile machine.

 Characterization of Materials

Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The nanocomposite scaffolds were investigated with FTIR spectra. All spectrums were taken at room temperature on a spectrometer with spectral range of 4000–400 cm⁻¹ and 4.0 cm⁻¹ resolution. Spectra were performed for PVA, Alginate, and PVA/Alginate scaffolds nanofibre containing 10% wt. BG. To evaluate the chemical composition changes of nanofibres the FTIR (Perkin-Elmer, UK) was used.

 Morphology of Electrospun Nanofibers Analysis

The surface morphology and diameter of the electrospun nanofibers were investigated with a scanning electron microscope (SEM; Philips XL30, Netherlands). Small sections of Al foil which coated with electrospun nanofibers was cut and sputter coated with gold to prevent electron charging. All of the samples were coated with gold (Au) using spraying, high vacuum and 40 kV accelerating voltage.

Inductive Coupled Plasma Atomic Emission Spectroscopy (ICP-OES) Analysis

The concentrations of Ca, Si, and P ions in SBF after soaking were determined using inductive coupled plasma atomic emission spectroscopy (ICP-AES) with Zaies (110394c).

RESULTS AND DISCUSSION

FTIR Analysis

FTIR spectra were also recorded for PVA and Alginate and PVA/Alginate nanocomposite (see Fig. 2). The characteristic band at 3442 cm⁻¹ was detected for the stretching frequency of --OH groups as shown in Fig. 2. Asymmetric and symmetric stretching vibrations for methylene groups appeared at around 2940 cm⁻¹. A significant band at 1500-1600 cm⁻¹ is attributed to –C=O group stretching vibrations. The strong peak at 1243 cm⁻¹ is attributed to stretching vibrations of C-O groups (Fig. 2). The bands around 2925 and 2854 cm⁻¹ 1 are assigned to asymmetric and the symmetric stretching to -CH₂ group, respectively. Two peaks at 1737 and 1434 cm⁻¹can be observed for to -CO stretching vibrations: -OH and -CH bending respectively. Heydari et al. [1] investigate a novel type of Gum and nanoclay bioceramic mixed (with PVA solutions. In their work the spectra of PVA/Gum/Nanoclay blend nanofibre, the absorption peak at 1617 cm⁻¹ was assigned to asymmetrical stretch of COO⁻ group which appeared at 1594 cm⁻¹ for Gum and shifted to 1618 cm⁻¹ for Gum/PVA nanofibre, suggesting that most of the carboxylic acid groups were associated with intramolecular hydrogen bonding.

The SEM and EDX micrograph of nanocomposite scaffolds in Fig 5 (a-d) shown that the density of fibers increased after 28 days of soaking in the SBF solution. Also, the nanocomposite scaffold containing 10% of BG had the cauliflower and bone-formation in the maximum of possible amount. The maximum amount of bone-like apatite formation is due to stable state of the BG nanoparticles powder in the SBF solution.

As it is seen in Fig 5, there are heavy and mass agglomerates particles that created in the nanocomposite scaffold with higher percentage of BG (see Fig. 5-d). The chemical stability of nanofiber increased with adding BG nanoparticles into the nanocomposite scaffold. It seem that that the agglomeration decreased by adding the BG nanoparticles. The result arises from the accumulation of brittle ceramic phases as a consequence of increasing BG amount.

Adding more than 5% BG leads to more pile of nanoparticles in polymer structure resulting in the reduction of mechanical features. SEM micrographs explain that the blend fibres have a shorter diameter pattern in the presence of more amount of BG. All the scaffold nanocomposite soaked in SBF solution were characterized by EDX analysis to recognize the presence of the elements on their surface (Fig. 5). EDX results on the large white tiny particle indicate that it include mainly oxygen, sodium, magnesium, phosphorus, chlorine, calcium, carbon and silicon. The carbon peak on the EDX analysis comes from the PVA as well as the Alginate. White tiny spots on the EDX maps represent the cite with maximum concentrations of the studied elements, and also proved the occupancy of apatite formation. Dissolution curves can be very helpful in this area that indicates the changes in weight of nanocomposite scaffold values versus immersion time in SBF (see Fig. 6). Also, the EDX analysis show there is amount of Na, P, Ca, and K in the tiny agglomerate scaffold sample. As the samples soaked in the SBF for long period of time, the rate of bioactivity decreased. The alteration in BG nanoparticles amount leads to higher AAS in the scaffold samples. The scaffold absorbs the calcium ions from the first week up to the last week. The highest rate of bioactivity dedicate to the sample with higher amount of BG nanoparticles. The research studies show that four scaffold nanocomposite PVA/Alginate/BG prepared by ELS technique. The surface characterization of the mineral phase of bone HA showed high ability to produce cauliflower and tiny agglomerate particles. It can be concluded, the formation of apatite on scaffolds surface must be considered [25-27].Here we try to calculate the amount of atomic absorption using calcium ions content examination. In the present case, the density of the pellet material is much higher as compared to that of the trabecular bone and upon foaming they may well match to further confirms this, the density of the foam materials was calculated and the average density of the foam scaffolds was found to be 0.92 g/cm³. Once present in the body, the material is expected to start degrading and will lead to further decrease in the density of the material due to the increase in the size and number of pores. Therefore, the density is low enough to mimic the trabecular bone whose density lies in the range of 0.09–0.75 g/cm³.