Polyurethane/58S bioglass nano ﬁ bers: synthesis, characterization, and in vitro evaluation

In this study, polyurethane nano ﬁ bers containing 5 and 10 wt% synthesized 58S bioglass were designed and fabricated by the electrospinning process. The physicochemical and mechanical properties and in vitro behavior of the sca ﬀ olds were evaluated by scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA), Fourier transform infrared spectroscopy (FTIR), contact angle, phosphate-bu ﬀ ered saline (PBS) uptake, in vitro bioactivity, MTT assay, and cellular response. The FTIR results showed an increase in the urethane bond between the OH (from BG) and NCO (from PU) groups with the increase in BG. When the BG amount increased from 5% to 10%, the contact angle decreased to approximately 20 (cid:1) and the PBS uptake of the sca ﬀ old increased because of the hydrophilic property of the BG particles. DMTA showed that the glass transition temperature started to shift slightly to higher temperatures from (cid:3) 17 (cid:1) C to (cid:3) 15 (cid:1) C. SEM and X-ray di ﬀ raction analysis depicted hydroxyapatite formation on the sca ﬀ olds upon immersion in SBF. Cell proliferation and viability also increased signi ﬁ cantly with the increase in BG. It is concluded that this composition provides a novel alternative for bone tissue engineering.


Introduction
Tissue engineering is an alternative treatment for repairing and regenerating damaged tissues. In hard tissue engineering, bioceramics, biopolymers, and their composites have been considered suitable candidates for bone tissue engineering. [1][2][3][4] Bioceramics, such as hydroxyapatite, tricalcium phosphate, glass-ceramics and bioglass (BG), have been used in different forms for bone tissue engineering. [5][6][7][8][9][10] They can be combined with biopolymers to improve the property of scaffolds, including their mechanical properties, biocompatibility, and bioactivity. 8,[11][12][13] BG powder has been synthesized using conventional melting methods and the sol-gel process. The sol-gel technique is an economical and technically simple procedure which has many advantages such as low crystallization temperature, high surface area and chemical homogeneity. 14,15 Several studies have shown that BG is angiogenic, 16 osteoconductive, 17,18 and osteoinductive. 19 BG also supports osteoblasts, which are vital for adhesion, growth, differentiation, and new bone formation in bone tissue engineering. 13,20 Studies demonstrated that bioglass-58S has high bioactivity, biodegradability and osteoconductivity. 21 Also recent research have shown degradation of bioglass can active gene expression and stimulate the production of growth factors. 22,23 In another point of view, polyurethane (PU) is one the most interesting biopolymers for bone tissue engineering because of its properties and relatively good biocompatibility. [24][25][26] Studies on PU and its potential application as a bone tissue material have increased because of its easy processability, biocompatibility, and mechanical properties. [24][25][26] To our knowledge, only a few studies on the development of PU/BG scaffolds for bone tissue engineering have been reported. [27][28][29] Different methods have been introduced to develop bone tissue engineering scaffolds, including salt leaching, 30 3D printing, 7,31 and electrospinning. 32 Recent studies have shown that electrospinning bers are suitable candidates due to it can provide many benets such as suitable conditions for the migration, and differentiation of bone tissue cells, wide range of nanobers and morphologies, cost-effectiveness and easy to set up. 32,33 In the present study, PU/BG scaffolds are prepared by the electrospinning method for the rst time to our knowledge. The prepared scaffolds were characterized in terms of physicochemical and mechanical properties. Moreover, apatite formation ability and in vitro biocompatibility were investigated.

Synthesis and characterization of bioactive glass nanoparticles
Bioactive glass based on the 58SiO 2 -33CaO-9P 2 O 5 system was synthesized by the sol-gel process. In brief, tetraethyl orthosilicate (Sigma-Aldrich, St. Louis, MO, USA) was added to the mixture of deionized water and hydrochloric acid (mole ratio of 1 : 8). Aer 30 min of mixing, triethyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) was added to the mixture and mixed for 20 min. Then, calcium nitrate (Merck, Darmstadt, Germany) was added and stirred for an additional 1 h. Thereaer, ammonia solution (1.0 mol L À1 ) was added to the acid sol, with vigorous stirring; then, the sol suddenly gelated. The synthesized gel was heated at 700 C for 3 h. The synthesized BG was analyzed by X-ray diffraction (XRD; XRD 3003 PTS), Fourier transform infrared spectroscopy (FTIR; Nexus 870), and transmission electron microscopy (TEM; EM208 Philips).

Electrospinning of scaffolds
Electrospinning was employed to fabricate random nanober scaffolds by using PU with and without BG. The polymer was dissolved in DCM/DMF (1 : 4, v/v) at a concentration of 10 wt%. Then, BG (5 and 10 wt%) was added to the solution dispersed with Dolapix CE64 (Zschimmer & Schwartz GmbH Co., Burgstädt, Germany) for 3 hours. Then, the PU (with and without BG) was electrospun by injecting the solution onto the covered aluminum foil collecting surface by using a syringe at a rate of 0.5 mL h À1 , with a distance of 18 cm from the needle tip. A 13 kV difference was applied across the syringe and the collecting surface. All solution preparations and electrospinning studies were conducted at room temperature. The scaffolds were investigated by scanning electron microscopy (SEM; KYKY-EM3200), FTIR (Thermo Nicolet Nexus 870), and contact angle goniometer (OCA 15 plus, Dataphysics). The apatite formation ability of the scaffolds was investigated aer immersion in SBF for different days. The structure and apatite formation ability of the scaffolds were monitored by XRD and SEM. In vitro evaluation was  conducted by cell culture and MTT assay. The cell attachment was observed by SEM.

Phase structure and morphological characterization
The synthesized BG powders and apatite formation of PU/BG scaffolds were analyzed by X-ray diffractometer (XRD 3003 PTS), with monochromatic Cu-Ka radiation (1.5418Å). The XRD diagrams were recorded in the interval 0 # 2q # 40 , with a step size of 0.02 every 1 s. The FTIR spectra were collected by the Thermo Nicolet spectrometer (Nexus 870) in the range of 400 cm À1 to 4000 cm À1 , and the spectral resolution was at a minimum of 4 cm À1 . TEM (EM208, Philips, Amsterdam, Netherlands) was used to investigate the morphology and the average particle size. Aer dispersing the powders in methanol with ultrasonication, the samples were collected on carboncoated gold grids operated at 200 keV. In terms of structure and morphology observation, the samples were sputter-coated with gold prior to scanning electron microscope (KYKY-EM3200) analysis.

Physical properties and mechanical analysis
The sessile drop method was employed to measure the contact angle by using the contact angle goniometer (OCA 15 plus, Dataphysics). The liquid droplet was deposited by a syringe pointed vertically down onto the sample, and the angle was determined by a high-resolution camera and analyzed by analysis soware. The phosphate-buffered saline (PBS) uptake of PU and PU/BG nanobers was evaluated through a simple method. Aer immersing the samples in PBS solutions in a 37 C shaking incubator for different days and changing the PBS solution every day, the samples were removed at 3, 6, 9 and 12 days. Aer drying, the samples were weighed. Then, PBS absorption (%) was calculated as follows: where W a and W b are the weights before and aer PBS soaking, respectively. Dynamic mechanical thermal analysis (DMTA) was conducted by using Teritec 2000 over a temperature range of À50 C to 200 C at a heating rate of 10 C min À1 and frequency of 1 Hz to investigate the effect of BG addition on the bulk material properties of the PU-based composites. The dimension of the samples was 30 Â 10 Â 1 mm 3 . The value of tan d (phase lag) and the elastic and loss moduli were recorded as a function of temperature for each sample.
In vitro studies L929 mouse broblasts were used to assess the biocompatibility of nanobers, and 4 Â 10 4 cells were used for each sample. The cells were enriched with Dulbecco's modied Eagle's medium supplemented with 4 mM glutamine, 10% fetal bovine serum, and 100 units per mL penicillin/ streptomycin and incubated at 37 C in a humidied atmosphere with 5% CO 2 . The cell morphologies were observed aer 24 and 48 h of cell culture. MTT assay was conducted to determine the cell proliferation and viability rates. Aer cell seeding on the nanobers and culturing for 48 h, 100 mL of MTT solution (5 mg mL À1 ) was added and incubated for 4 h at 37 C in a humid atmosphere with 5% CO 2 . Then, the MTT solution was removed, the samples were washed with PBS, and 0.5 mL of dimethyl sulfoxide was added. Finally, the viable cells in the solution were quantied by using a scanning multiwell spectrophotometer (ELISA Microplate Readers, BioTek) at 540 nm. The polystyrene surface of the cell culture plates was used as the control. Samples were prepared by xing cells with 2.5% glutaraldehyde, rinsing with PBS, and dehydrating in graduated ethanol from 50% to 100% in steps of 10% for 5 min each to observe cell adherence and its morphology on nano-bers by SEM.

Statistical analysis
All the measured values were presented as the mean AE standard deviation of at least ve experiments. The signicance between the mean values was calculated by using one-way analysis of variance and Student's t test. Differences were considered signicant at p # 0.05.

Results and discussion
The XRD and TEM data for BG are shown in Fig. 1a and b, respectively. BG exhibited an amorphous phase, and the size of the powders was less than 50 nm. The FTIR spectra of 58S bioactive glass, PU, PU with 5 wt% bioglass (PU5BG), and PU with 10 wt% bioglass (PU10BG) are shown in Fig. 2. The BG spectrum revealed the presence of Si-O and Si-O-Si peaks at 462 cm À1 and 725 cm À1 , which can be assigned to rocking and bending vibrations, respectively, 34 and 1024 cm À1 attributed to the phosphate group. 28 The shoulder band at 821 and 1565 cm À1 can be attributed to the bending of the P-O vibrational    PU and hydroxyl group from BG 6,42 and the chemical linkage between hydroxyl groups of BG and PU, which causes the disappearance of the peaks at 3550 cm À1 corresponding to the -OH groups in BG. 6,41 The storage modulus and glass transition temperature (T g ) of the prepared samples were investigated by DMTA. DMTA detects the type of transitions and relaxations that are related to the structure and morphology of the samples. Glass transition is identied by the decrease in storage modulus and the presence of prominent peaks in tan d. The DMTA thermographs of the unmodied PU and PU10BG composite are shown in Fig. 3A and B, respectively. The T g of the unmodied PU is approximately À20 C, but started to shi slightly to higher temperatures from À20 C to À14 C and broadened aer being modied with BG. The storage modulus also increased to a higher value in PU modied with BG than pure PU. The presence of inorganic phase BG and its increase to 10% increases the T g by hindering the molecular and segment rotation with reference to the polymer. This effect also reduces the dipole interaction potential and makes the polymer mechanically stronger.
As shown in Fig. 4A, PU bers were produced smoothly, continuously, and bead-free. The ber diameter histogram which determines by Image J soware shows in Fig. 4D and nanobers are mainly ranged below 200 nm. Fig. 4B and C show the SEM of PU5BG and PU10BG as-spun nanobers, respectively. As shown in Fig. 4A-C, the morphology of the nanobers did not change with the addition of BG, which was in accordance with a previous research by Khan et al. 38 Previous studies have shown that the size of PU nanobers depends on solution viscosity. With the addition of nanoparticles to the solution, the viscosity increased because of the linkage between PU and nanoparticles or the increase in molecular entanglement. 38 Electrospun nanobrous matrices show morphological similarities to the natural extracellular matrix and are characterized by high surface-to-volume ratio, high porosity, and variable pore size distribution, exhibiting properties that can modulate the cellular behavior. 33 Several biopolymers have been electrospun to produce nanobrous scaffolds for cartilage and bone tissue engineering. 43,44 Many bioceramics have been incorporated into biopolymers, and their mineralization, cell adhesion, proliferation, and differentiation have been investigated for use as bone substitutes. 45 Given the degradation of biopolymers, the environment would be acidic, which is unsuitable for the cells. Using bioceramics is a suitable method of buffering this environment. 9,46 Also adding bioceramics to biopolymers can also increase the mechanical properties of the scaffolds 47,48 and prevent the mechanical degradation of polymers. 9 In the present study, bioactive glass nanoparticles were incorporated into PU nanober to form the nanocomposite nanobrous web, and its bioactivity was investigated in simulated body uid (SBF). A signicant characteristic of bioactive materials is their ability to bond with bone through hydroxyapatite formation on their surface. 49 Previously, the authors observed that the BG-added polymers showed full coverage of the surface with apatite    precipitates, biocompatibility, cell growth, and differentiation. 48,50,51 The mechanical properties of the composites were also improved. 28 Fig. 5 shows the SEM of the bers aer SBF immersion for 3, 7, and 14 days under different magnications. This morphology is typical of hydroxyapatite, which has been reported to grow on the surface of bioactive glass/polymer composite scaffold aer incubation in SBF. The difference between the morphology of the surfaces can be evidently observed. Apatite covers the surfaces with BG, and the formation of apatite increased with the increase in the BG amount, as conrmed by XRD. The XRD of the samples aer soaking in SBF for seven days showed increase in the formation of the apatite phase with the increase in the BG content (Fig. 6). Hydroxyapatite formation due to the reactivity of BG will stimulate a physiologically relevant environment for the cells to adhere and proliferate. Radev et al. 35 showed that nanohydroxyapatite formed on the surface of PU/ 85S composite aer seven days of incubation in SBF and proposed this composite for bone tissue engineering application. The deposition of a hydroxyapatite layer on the surface of the bioactive materials soaked in SBF involves the mechanism proposed by Hench. 50 The results indicated the formation of HA that increased gradually with time, consistent with previous research on the immersion of BG and PU/BG composites in SBF. 28,[52][53][54] Pores with diameters larger than 150 mm are necessary to accelerate internal mineralized bone formation, whereas pores with diameters smaller than 10 mm inhibit cellular ingrowth. Pores with diameters between 15 mm and 150 mm are suitable for brovascular colonization, osteoid growth, cellular penetration, and proliferation. 55 Fig. 4 shows that the electrospinning method was successful in generating these types of pores.
The contact angle test results (Fig. 7) showed that the pure PU scaffold had the highest contact angle value at 105 AE 3.2, which decreased from 92 AE 2.1 to 72 AE 1.8 with the increase in BG content from 5% to 10%. Fig. 8 shows that scaffolds with a higher BG amount exhibited higher PBS uptake because of BG hydrophilicity. 56 When the scaffold is pure PU, the PBS uptake content increased to 16% aer six days, which increased to 33% with the increase in BG to the highest amount. These last two tests conrmed the hydrophilicity of BG. The hydrophilic property of the scaffolds can facilitate cellular nutrient supply, cell adhesion and growth, and waste removal and maintain differentiated phenotypic expression. 28,57 The hydrophilicity of BG can affect the glass/polymer interface, degradation rate, and pH buffering effect. 56,58 Good adhesion between ller and polymer can be obtained by intimate contact between phases, which is a result of suitable wettability. 59 In this study, L929 mouse broblasts cells were seeded on the surface of the scaffolds and cultured for 24 and 48 h. Fig. 9 shows the cell morphology aer 24 and 48 h for the control, PU, PU5BG, and PU10BG groups. Aer culturing for 48 h, the cell morphology was observed by SEM and shown in Fig. 10 with two magnications. The cells could grow, adhere, and stretch on the surfaces of all scaffolds. The MTT assay showed a low death rate because of the biocompatibility of the scaffold materials, which was enhanced with the increase in the BG amount (Fig. 11). This result indicates that the BG-containing scaffolds may have accelerated cell proliferation. The area covered with cells on the scaffolds increased with the increase in BG concentration. Ionic dissolution in bioactive glasses plays an essential role in such osteoconductivity, angiogenesis, 60 and cellular processes as bone cell phenotypes, bone cell differentiation, and gene expression. [61][62][63][64] For example, the release of Ca 2+ depresses osteoclast-mediated bone resorption and addresses osteoblastic proliferation and the presence of a high concentration of phosphorus induces osteoblast apoptosis. 41,42 Based on our observations, the incorporation of 58S BG in PU has potential for bone tissue engineering because of its bioactivity, cellular response, and wettability. However, more investigations are necessary to conrm our ndings.

Conclusion
In this study, highly in vitro bioactive and biocompatible materials, in the system of SiO 2 -CaO-P 2 O 5 , have been obtained by the sol-gel technique. Moreover, a novel porous scaffold based on PU and 58S BG was fabricated by electrospinning. This study shows that covalently linked PU/BG composite and the nanobres of this composite were successfully collected by the electrospinning process and their diameter is below 200 nm. The morphological appearance of the nanobres was smooth and uniform. The effect of different amounts of BG (5 and 10 wt%) on the physical and chemical properties, bioactivity, wettability, PBS uptake, cellular response, and MTT assay was investigated. The incorporation of the BG in the PU/BG scaffold signicantly enhanced the bioactivity, biocompatibility, hydrophilicity and storage modulus of composite. L929 broblast cells adhered to and grew well on the bioglass-added PU scaffold, with a higher level of growth than on the pure PU. In brief, the results showed that the prepared scaffolds represent an interesting candidate for bone tissue engineering because of their ease of fabrication and suitable properties.
Malaya and Iranian Council for Stem Cell Sciences and Technologies for nancial supports. The technical assistance of Dr Sumit Pramanik for the Image J analysis is appreciated.