A novel approach to fabricate silk nanofibers containing hydroxyapatite nanoparticles using a three-way stopcock connector
© Sheikh et al.; licensee Springer. 2013
Received: 13 May 2013
Accepted: 18 June 2013
Published: 1 July 2013
Electrospinning technique is commonly used to produce micro- and/or nanofibers, which utilizes electrical forces to produce polymeric fibers with diameters ranging from several micrometers down to few nanometers. Desirably, electrospun materials provide highly porous structure and appropriate pore size for initial cell attachment and proliferation and thereby enable the exchange of nutrients. Composite nanofibers consisting of silk and hydroxyapatite nanoparticles (HAp) (NPs) had been considered as an excellent choice due to their efficient biocompatibility and bone-mimicking properties. To prepare these nanofiber composites, it requires the use of acidic solutions which have serious consequences on the nature of both silk and HAp NPs. It is ideal to create these nanofibers using aqueous solutions in which the physicochemical nature of both materials can be retained. However, to create those nanofibers is often difficult to obtain because of the fact that aqueous solutions of silk and HAp NPs can precipitate before they can be ejected into fibers during the electrospinning process. In this work, we had successfully used a three-way stopcock connector to mix the two different solutions, and very shortly, this solution is ejected out to form nanofibers due to electric fields. Different blend ratios consisting HAp NPs had been electrospun into nanofibers. The physicochemical aspects of fabricated nanofiber had been characterized by different state of techniques like that of FE-SEM, EDS, TEM, TEM-EDS, TGA, FT-IR, and XRD. These characterization techniques revealed that HAp NPs can be easily introduced in silk nanofibers using a stopcock connector, and this method favorably preserves the intact nature of silk fibroin and HAp NPs. Moreover, nanofibers obtained by this strategy were tested for cell toxicity and cell attachment studies using NIH 3 T3 fibroblasts which indicated non-toxic behavior and good attachment of cells upon incubation in the presence of nanofibers.
Recent advances in tissue-engineering techniques had enabled scientists in fabricating the novel scaffolds with multi-functional properties to overcome the problems faced in the existing one. The 2D and/or 3D scaffolds used for tissue-engineering applications had greatly influenced the present scenario for scaffold construction. Although there are lots of advances made in tissue-engineering, the scientific community is still facing a major challenge to select the perfect strategy and choice of materials while considering the fabrication of scaffolds. The most uniquely used biopolymer made from silk fibroin proteins are obtained from silkworms and had a long history of applications in the human body as sutures. Silk fibroin contains peptides composed of RGD sequences that can promote cell adhesion, migration, and proliferation [1, 2]. These attractive properties of silk fibroin are particularly useful for selecting them as a material of choice for tissue-engineering applications . The efficient biocompatibility, minimal inflammatory response to host tissue, relative slow biodegradation rates compared with other materials, and easy availability from sericulture industry make the silk fibroin a desirable candidate for various medical applications .
On the other hand, hydroxyapatite (HAp) is a major solid component of the human bone which can be used as a vital implant due to its excellent biocompatibility, bioactivity, non-immunogenicity, non-inflammatory behavior, and osteoconductive nature . However, the loose and particulate nature of HAp seriously hampers its use in any tissue-engineering applications . In order to utilize the HAp for tissue regeneration especially in the form of scaffolds, it must meet most of the desired requirements, such as desirable mechanical support to sustain the pressure surrounding the host tissues and simultaneously should provide high porosity. For this reason, HAp is often blended with other supporting materials to make its practical utility possible. Desirably, a suitable material is selected to blend with HAp for the facilitation of proper cell seeding and diffusion of nutrients for the healthy growth of cells during the initial period of implant which is considered as crucial .
Among available methods, to create a suitable scaffold in which these biologically important materials can be incorporated is the electrospinning technique, which had emerged as a versatile technique to convert biologically significant polymers into nanofibers, so as to use them as potential candidate for tissue-engineering [8–12]. The unique characteristics such as very high surface area-to-volume ratio, high porosity, and capability to mimic the extracellular matrix (ECM) present in the human body had created a special attention on nanofibers produced by the electrospinning technique. Due to these features, electrospun nanofibers had been used as potential candidates for many biomedical applications, such as in drug delivery, wound dressing, and scaffolds for tissue engineering [10–12]. This technique can produce micro- or nanofiber of various polymers in the form of non-woven mats which are similar to the structure present in the natural ECM, which is vital for initial cell adhesion, as a biomimicking factor of cells [13–16]. In this connection, many biodegradable synthetic and/or natural polymers have been electrospun into nanofibers to be used as scaffolds for various tissue repair and regeneration such as bone, cartilage, vascular blood, nerve, skin, and bladder [13–17].
The use of electrospinning to fabricate the silk-based nanofibers and HAp nanoparticles (NPs) had been exploited to create 2D scaffolds. For instance, efforts to modify silk fibroin nanofibers to attribute properties of HAp was done by soaking in stimulated body fluid (SBF) by Kim et al., and this similar mineralization approach had been also frequently used by other researchers [18, 19]. However, this soaking method by SBF results in superficial attachment of HAp NPs on nanofibers. In order to have HAp NPs with strong bonding with nanofibers, the use of freeze-dried silk crystals and strong chemicals had been adapted to create nanofibers containing HAp NPs [20, 21]. However, it is noteworthy to mention that the use of strong chemicals in that case further restricts the biocompatibility aspect of nanofibers. Therefore, an alternative strategy is needed to fabricate the silk fibroin nanofibers having the features of HAp NPs. The use of aqueous silk/HAp blend solutions can be considered as an ideal way to form nanofibers. By doing that, HAp NPs will be strongly fixed to nanofibers, and intact nature of silk/HAp can be preserved without using toxic chemicals. However, due to large functional groups present in silk, HAp NPs can lead to form a bond due to abundant hydroxyl groups present in these biologically important materials and make it difficult to electrospun [22, 23].
In this work, for the first time, we presented the use of aqueous regenerated silk fibroin solution blended with HAp NPs using a three-way stopcock connector. In our system, the aqueous silk solution and HAp NPs colloidal suspension combine together at the center of the three-way connector for a short time without giving enough time to precipitate, and this blend solution is immediately ejected out to form nanofibers. Different weight ratios of 10%, 30%, and 50% of HAp NPs were used as blend solution to electrospun nanofibers. The obtained nanofibers were characterized for various psychochemical characterizations, and interaction of these nanofibers with fibroblasts was done to study the cell toxicity and cell attachment of nanofibers incorporated with HAp NPs.
Silkworm cocoons were obtained from the Rural Development Administration (Suwon, Republic of Korea). Poly(ethylene oxide) (PEO) with an average molecular weight of 200,000 (Sigma-Aldrich, St. Louis, USA) was used as sacrificial polymer to electrospun silk solution and to make HAp/PEO colloid solutions. HAp rod-shaped NPs measuring 30 to 60 nm were obtained from Dae Jung, Siheung, Gyeonggi, Korea. NIH 3 T3 fibroblasts were purchased from ATCC (Manassas, VA, USA.). Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fatal bovine serum, cocktail of 1% penicillin-streptomycin, Trypsin, were obtained from Welgene, Fresh Media™ (Dalseogu, Daegu, Korea). Trypan Blue Stain 0.4% was obtained from Gibco® (Life Technologies Corporation, Gaithersburg, MD, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent used to check the cell viability was purchased from Duchefabiochemie, Haarlem, The Netherlands. Dimethyl sulfoxide (DMSO) with high purity grade of 99.9% was acquired from Sigma-Aldrich. Tissue culture flasks and microplates for cell seeding and growth were purchased from BD Falcon™, Winston-Salem, NC, USA and SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea.
Variable pressure field emission scanning electron microscope (FE-SEM) EVO® LS10 equipped with energy-dispersive X-ray spectroscopy (EDS) obtained from Carl Zeiss SMT., Ltd., Oberkochen, Germany, was used to investigate the morphology and elemental detection of nanofibers. Before viewing, the samples were pasted on a carbon tape and sputter-coated using a thin layer of gold palladium for 120 s for two consecutive cycles at 45 mA with the Ion Sputter 1010, Hitachi, Chiyoda-ku, Japan. After sample coating, the micrographs from each samples were taken at an accelerating voltage of 2 KV and with magnifications of 15 K. The EDS images were captured at an accelerating voltage of 10 KV and with magnifications of 15 K. The average nanofiber diameters were calculated using the software Innerview 2.0, Dong, Bundang Daeduk Plaza, Korea, after measuring 100 diameters per sample from FE-SEM images. Transmission electron microscopy (TEM) was done by JEOL JEM-2200FS operating at 200 KV, JEOL Ltd., Akishima-shi, Japan. The samples for TEM were prepared by dispersing 10 mg of nanofibers in 200 μl of ethanol and subsequently dispersed by bath sonicator using locally supplied ultrasonic cleaner (60 kHz, Shenzhen Codyson Electrical Co., Ltd., Shenzhen, Guangdong, China) for 120 s. After dispersing the nanofibers, 20 μl of dispersion was pipetted out by micropipette and carefully poured on 200 mesh copper grid. The extra solution was removed using Kimwipes supplied by Kimberly-Clark Professional, GA, USA, and the grid was allowed to dry overnight at room temperature. Information about the phases and crystallinity was obtained using PANalytical diffractometer (HR-XRD, X’pert-pro MPD, Almelo, The Netherlands) with Cu, Cr (λ = 1.540 A) radiation over Bragg angle ranging from 10° to 60°. To identify the vibrations caused due to functional groups in nanofibers, Fourier transform infrared spectroscopy (FT-IR) analysis was done using BIO-RAD (Cambridge, MA, USA). The samples were directly loaded on ATR window, and spectra were collected using Excaliber Series by averaging 32 scans with the resolution of 4 cm−1. The thermal analysis of the synthesized nanofibers was carried out with a thermal analysis system, (TA Instruments, New Castle, DE, USA) by ramping the samples at 10°C/min, and heating was started from 30°C to 700°C. Heating was followed under a continuous nitrogen purge of 100 mL/min, and spectra were collected using Q600 Software (TA Instruments). For checking the cell attachment on nanofibers by FE-SEM, the images were captured with an accelerating voltage of 3 KV with magnifications of 1 K.
Preparation of aqueous regenerated silk solutions
The aqueous silk solutions to be used for electrospinning were prepared by the following procedure. Firstly, degumming was achieved by cutting Bombyx mori cocoons into suitable pieces and were boiled in 0.02 M Na2CO3 for an hour and subsequently washed with de-ionized water (2 to 3 times) to remove the unbound sericin. Later on, the samples were dried at room temperature for 1 day. After drying, 60 g of degummed silk was dissolved in ternary solvent composed of CaCl2/Ethanol/H2O (32/26/42, wt/wt/wt) at 98°C for 40 min in round-bottomed flasks. Following this, protein mixture was filtered through miracloth (Calbiochem, San Diego, CA, USA) to remove small aggregates. Furthermore, this solution was dialyzed against deionized water using a dialysis tubing with molecular weight cutoff 12,000 to 14,000 Da (Spectra/Por®, Rancho Dominguez, CA, USA) for 3 days, and water was exchanged once a day. The yielding aqueous silk fibroin solution was calculated to be 8 wt.% (which was determined by weighing the remaining solid weight after drying). Finally, the aqueous silk fibroin solutions were stored in a refrigerator and used within 15 days of time to avoid denaturation and/or precipitation.
Nature of used HAp NPs
Polymeric solution preparation for electrospinning
For preparing solution to electrospun pristine silk nanofibers, 20 ml of 8 wt.% of aqueous silk solution was removed from the refrigerator. To give appropriate viscosity to this solution, so as to have proper bending instability for fiber formation, 4 ml of previously prepared 30 wt.% PEO solution was added as a ‘sacrificial polymer.’ The resultant blend solutions were loaded in syringes and used for electrospinning. For preparing solutions to fabricate silk fibroin nanofibers containing HAp NPs, a stepwise methodology was adopted. On one hand, silk solution was prepared in the same way as mentioned for the preparation of pristine silk nanofibers and subsequently loaded in syringes. On the other hand, PEO/HAp colloidal solution was prepared by adding 2 g of PEO in 20 ml of 0.001 molar solution of phosphate buffer saline (PBS), and this solution was mixed well to solubilize. To this solution, HAp NPs were added to give the final concentration of 10%, 30%, and 50% HAp with respect to 8% of aqueous silk fibroin solution. After adding HAp NPs in PEO solution, the HAp NPs were agitated using an ultrahigh sonication device. This was achieved using Sonics Vibra-cell model VCX 750, Newtown, CT, USA, operating at 20 kHz with an amplitude of 20%. The ultrasonic agitation was allowed to continue for a period of 1 min. After complete sonication, the samples were viewed as homogeneously dispersed and well stabled without being precipitated at the bottom. Further on, these dispersed HAp/PEO solutions were filled into the syringes to be used for electrospinning.
Cell viability and cell attachment studies
The frozen ampules of NIH 3 T3 fibroblasts removed from liquid nitrogen tank were incubated at 37°C for 1 to 2 min to form a semisolid suspension. The cells from these ampules were taken out and added with fresh media, centrifuged to get cell debris, and enriched with fresh media allowed to incubate at 37°C for 3 days for the completion of the first subculture. In this study, cells were used after two subcultures to check the cell viability, and cell attachment with renewal of culture media was done after 3 days. The nanofiber samples used for checking cell viability and cell attachment studies were pierced into disk shapes using biopsy punchers (Kasco, Keys Cutaneous Punch, Sialkot, Pakistan) forming 6-mm round disks, giving it an appropriate diameter to fit in a 96 well plate. Each nanofiber disk was sterilized by dipping it in 70% ethanol in 6-well plate for 30 min. The excess of ethanol on nanofibers after sterilization was rinsed by dipping the samples in 10 mL of DMEM. Further on, the nanofiber samples were transferred on 96-well plates in triplicates. A 100 μl of cell suspension containing 25,000 cells/mL was counted using cell counting method, and the cells were carefully seeded over the top of sterilized nanofiber disks in the 96-well plate. The seeded scaffolds were incubated at 37°C for 30 min to allow cell adhesion. Following this, 100 μl of fresh medium was added in each well, and the plates were incubated in a humidified incubator with 5% CO2 environment at 37°C for 1, 2, and 3 days. The cell viability was evaluated by MTT reduction assay. After desired days of incubation, the media from 96-well were suctioned out and treated with 200 μl of the MTT solution, by mixing the contents by side-tapping, and further on, these plates were incubated at 37°C for 2 h. After incubation, MTT solution was suctioned out and added with 200 μl of DMSO, which was subsequently rocked to form purplish blue-colored formazan solution. The solubilized formazan appearing from each well were transferred to fresh wells of 96-well plate for spectrophotometric analysis at 540 nm in an ELISA microplate reader (Molecular Devices, SpectraMax® Plus 384, Sunnyvale, CA, USA). The cell viability was obtained by comparing the absorbance of cells cultured on the nanofiber scaffolds to that of the control well containing DMSO. For cell checking attachment on nanofibers, the cells were allowed to grow for 3 and 12 days’ time, and media was changed after every 3 days. To check the cell morphology, cell fixation and dehydration was done by rinsing the samples twice with PBS followed by fixation with a 2.5 vol.% glutaraldehyde solution for 4 h. After cell fixation, the samples were rinsed with PBS and then dehydrated with graded concentrations of ethanol (20 vol.%, 30 vol.%, 40 vol.%, 50 vol.%, 70 vol.%, and 100 vol.% ethanol) for 10 min each. Finally, the samples were kept overnight in a vacuum oven and observed in FE-SEM to determine cell attachment. The samples for FE-SEM were coated by keeping the same conditions as described previously in the ‘Characterization’ section. However, the micrographs of each sample were taken at an accelerating voltage of 2 KV and with magnifications of 15 K.
Results and discussions
In conclusion, a highly trustable technique which employs the use of stopcock connector can be used to electrospun a blend solution of fibroin and HAp together in aqueous solutions, which is impossible if simple mixing procedure is followed. Without the use of any toxic chemical, this technique can yield nanofibers with desirable properties. The FE-SEM and TEM techniques can be used to figure out the location of HAp in nanofibers and simultaneously support the use of stopcock connector to electrospun silk fibroin and HAp NPs. Fourier transform infrared spectroscopy analysis indicated the chemical interaction occurring between HAp NPs and silk fibroin, which resulted in the transformation of random coil to β-sheet confirmation of silk fibroin. It can also be concluded that HAp NPs enhanced the β-sheet conformation of fibroin and resulted in the improvement of the properties of nanofibers. Generally, it is believed that the presence of highly favorable components likeHAp could improve the cell attachment; however, in our case, we find that the attachment of fibroblast on nanofibers is independent to the presence of HAp. Moreover, the cell viability of nanofibers can be improved by this technique.
This research was supported by Hallym University Research Fund and the Biogreen 21 program, grant PJ009051062013, Rural Development Administration, Republic of Korea.
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