Interaction of magnetic cobalt based titanium dioxide nanofibers with muscle cells: in vitro cytotoxicity evaluation
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- Amna, T., Hassan, M.S., Khil, M. et al. J Sol-Gel Sci Technol (2014) 69: 338. doi:10.1007/s10971-013-3222-3
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Titanium dioxide and magnetic cobalt based materials are one of the most attractive materials for investigation due to their dramatic photocatalytic, optical, biomedical, magnetic and electrical applications. However, there is limited or no information about the possible impact of cobalt based titanium dioxide (Co-doped TiO2) nanofibers on muscle cells. This study focuses on the interaction of magnetic cobalt based titanium dioxide nanofibers with C2C12 cell line. C2C12 mouse myoblasts were used to evaluate the beneficial/or toxicological effects of Co-doped TiO2 on cells. The effects of Co-doped TiO2 nanofibers on the morphology, cytotoxicity and adhesion ability of C2C12 cells, as well as on the cell death were evaluated. To examine the in vitro cytotoxicity, mouse myoblast C2C12 cells were treated with different concentrations of synthesized Co-doped TiO2 nanofibers and the viability of cells was analyzed by cell counting Kit-8 assay at regular time intervals. The morphological features of the cells were examined by microscopy and cell attachment with nanofibers was observed by scanning electron microscopy respectively. Experiments indicate that the mouse myoblast cells could attach to the nanofibers after being cultured. Cell viability was determined as a function of incubation time; with increasing concentration of Co-doped TiO2, the cell viability decreased. Thus from the obtained results it was concluded that Co-doped TiO2 nanofibers could support cell adhesion and growth of myoblast cells, however the cell compatibility decreases with high doses and after sustained exposure.
KeywordsElectrospinningC2C12Co-doped TiO2ScaffoldsTissue engineering
Tissue engineering is an interdisciplinary science that intends to design and fabricate the artificial implants for in vivo tissue regeneration . Recently, hybrid or/composite nanomaterials have emerged as ideal multifunctional nanomedicine platforms for diverse remedial approaches. Hybrid materials generally possess novel hybrid characteristics. Each component of the hybrid/or composite has a specially designed function to enhance the overall effectiveness of the system. The interactions between cells and biomaterial surfaces play a significant part in regulating the physiological behavior (such as attachment, spreading, migration, proliferation, and differentiation) of cells [2, 3]. As a result, efforts have been made to control cellular responses by making adjustments in various parameters, for instance; the chemical composition, topography and 3-dimensional geometry of biomaterial substrates [4–6]. However; designing scaffolds for tissue engineering with ability to modulate morphogenesis and regeneration in implanted cells is crucial to achieve functionally active engineered tissues . It is well established that the living cells reside within the microenvironment made up of extracellular matrix (ECM) and exploitation of features of native ECM can be beneficial for regenerative cells. Recently, electrospun nanofibrous scaffolds have received great attention in tissue engineering because of their amazing properties such as tunable porosities, large surface area, and comparable structures to natural ECMs. In the present study we have evaluated the effects of Co-doped TiO2 nanofibers on the morphology, cytotoxicity, adhesion ability of C2C12 cells, as well as on cell death. There is limited information about the possible impact of Co-doped TiO2 nanofibers on human health or the environment. Furthermore, there are no studies in the literature investigating the toxic effects of Co-doped TiO2 nanofibers to C2C12 cells. To our knowledge, this is the first study in which C2C12 mouse myoblasts were used to evaluate the beneficial/toxicological effects of Co-doped TiO2 on cells. Nevertheless; previously  used electrospinning technique to fabricate aligned nylon 6/6 fiber scaffolds as ECM for C2C12 cells. They cultured C2C12 cells on the aligned fiber scaffolds and it was observed that C2C12 were organized accordingly to the fiber direction forming a continuous sheath, and differentiated into myotubes . Likewise, in another study, the elongation and alignment of C2C12 myoblasts and myotubes have also been shown in oriented small diameter (10–15 nm) carbon nanowhiskers . Whereas  fabricated nanofibrous and micropatterned polymers by electrospinning as cell culture substrates to guide the morphogenesis of muscular tissue. The nanoscale and microscale topographic features regulate cell and cytoskeleton alignment, myotube assembly, and myoblast proliferation. It was also reported that the nanofibers promoted the longer myotube formation and inhibited cell proliferation compared to flat controls and micropatterned substrates . Despite some studies about the interaction between C2C12 cells and nanotextured materials, however to our knowledge, the interactions between C2C12 cell lines and magnetic cobalt based titanium dioxide composite nanofibers have not been studied until now. To this end, the present study investigates the cytotoxicity of fabricated nanofibers against C2C12 cells using CCK-8 viability test. In the present study we have employed the electrospinning technique to produce nanotextured magnetic cobalt based titanium dioxide composite nanofibers. It has been established that the nanotextured morphology is accountable for modulating cell proliferation, migration, differentiation and apoptosis, as well as provide a set-up that can facilitate nutrient transport and diffusion. Ti and its alloys have long been used as implantable biomaterials because they possess excellent mechanical properties  which are adequate for transferring stress between implant and bone. Also they exhibit a surface native oxide layer (TiO2) that is resistant to corrosion. Moreover, they are biocompatible and bioactively react with native human plasma and tissue. Already TiO2 has been proven to be promising biocompatible materials and has been used as a bioactive ceramic [12–15] due to its novel characteristics. Previously, TiO2 scaffolds were implanted in rats for several weeks without any signs of inflammatory responses . Titanium and its alloys have been used extensively to fabricate implantable devices such as artificial blood vessels, joint prostheses, fracture fixation devices and dental implants . On the other hand, magnetic nanoparticles (NPs) have also many potential applications in biological and medical area. Cobalt is a class of ferromagnetic material with high magnetic moment density (160 emu/g or 1,422 emu/cm3) and is magnetically soft. The cobalt oxide (CoO and Co3O4) based materials possess remarkable optical, electrical and magnetic properties and are therefore commonly used for photocatalysis and electromagnetic applications [18, 19]. The composites Micro and nano-fabrication techniques can be used to generate scaffolds which can imitate native tissue (ECM) properties (such as promote cell attachment and clinically assist in the healing and regeneration process). Electrospinning is a material processing technique that produces fibers with nanoscale range . In the electrospinning process, a charged polymer jet is pulled out by applying electrostatic force and these jets at first extends in a straight line and then experience the whipping motion while travelling from the spinneret to collector and ultimately get deposited in the form of a non-woven fiber mat. Electrospinning is an easy way to control the morphology of fibers and the obtained fibers possess desirable characteristics. Considering the unique properties of titanium and cobalt, we have been using electrospinning technique in the present study to successfully fabricate Co doped TiO2 nanofibers.
2 Materials and methods
Poly(vinyl acetate) (PVAc, Mw = 500,000) and cobalt nitrate hexahydrate, (Co(NO3)2·6H2O) were purchased from Sigma-Aldrich, USA. N,N-dimethylformamide (DMF) and Titanium isopropoxide (TIP, 98.0 assay) were purchased from Showa and Junsei Co. Ltd., Japan respectively. Myoblast C2C12 cells (ATCC-CRL 1772) and cell counting Kit-8 (CCK-8), were purchased from American Type Culture Collection (ATCC) and from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) respectively. Dulbecco’s modified Eagle medium (DMEM) from Gibco® life technologies, Grand Island, NY, USA and laboratory wares were purchased from Falcon Labware (Becton–Dickinson, Franklin Lakes, NJ, USA) respectively.
2.2 Electrospinning and characterization
The fabrication of Co-doped TiO2 nanofibers was carried out by simple electrospinning method. Briefly, PVAc (18 wt%) solution was prepared by dissolving PVAc in DMF under magnetic stirring (~6 h) at room temperature. Few drops of acetic acid were added to 5 g of TIP till the solution turns out to be transparent. An alcoholic solution of Co(NO3)3·6H2O (5 wt%) was added into the TIP solution. Subsequently, 6 g of PVAc solution was slowly mixed with the aforementioned solution and stirred vigorously. The resulting sol–gel was transferred into a 10 ml syringe. A voltage of 15 kV was applied and the distance between the tip of the needle and collector was fixed at 18 cm. The as-spun nanofibers were initially dried at 80 °C for 24 h under vacuum and then calcined in air at 500 °C with heating rate of 2 °C/min for 2 h. The XRD pattern of nanofibers was recorded on a Rigaku/Max-3A X-ray diffractometer (XRD, Rigaku Co., Japan) with Cu Kα radiation (λ = 1.540 Å) over Bragg angles ranging from 20 to 80o and the operating voltage and current were maintained at 30 kV and 40 mA, respectively. The chemical composition of synthesized nanofibers was analyzed by energy dispersive X-ray spectrometer (EDX) and distribution of elements was measured using electron probe microanalysis (EPMA). To examine the microstructure, the powdered samples were uniformly sprayed on carbon tape and Pt coating was applied for 10 s. The images were acquired at various magnifications by scanning electron microscopy (SEM-S-7400, Hitachi High technologies, Tokyo, Japan). The detailed microscopic features of doped nanofibers were analyzed by transmission electron microscopy (TEM-H-7650 Hitachi High technologies, Tokyo, Japan).
2.3 Cell culture and cytotoxicity assay
The C2C12 cells were grown in 75 cm2 culture flask (Bedford, MA, USA) to get the enough cell count for cell seeding and subculture. Briefly, C2C12 cells were cultured in DMEM, (pH 7.4) containing 10 % fetal bovine serum and 1 % penicillin–streptomycin solution. Cells were grown and maintained in a humidified incubator at 37 °C with 5 % CO2 and 95 % air environment. The cell density of 1 × 104 cells/well was seeded in a 96-well plate (Becton Dickson and Company, Franklin Lakes, NJ) and allowed to attach and grow in wells overnight before treatment with magnetic cobalt based titanium dioxide nanofibers. The test suspension of Co-TiO2 was prepared using the culture media and dispersed by using a sonicator to prevent aggregation. The Co-TiO2 composite solution was sterilized under UV before use. When C2C12 reached ~60 % confluence, cells were treated with different concentrations (0, 5, 10, 15 and 20 μg/ml) of Co-TiO2 nanofibers for a specific time (24, 48, and 72 h) duration. Untreated cells were also run under identical conditions and served as control. The exhausted media were replaced with fresh DMEM medium at regular intervals throughout the incubation period.
The CCK-8 assay was performed to check the cell viability in the presence of Co-TiO2 nanofibers. In brief, media from the microplates was taken out after specified incubation and replaced with fresh media in which 10 μl of water-soluble tetrazolium-8 solution in each well was added and incubated for 4 h at 37 °C according to the manufacturer’s instructions. At the end of the experiment, absorbance was measured at 450 nm for each well by a microplate spectrophotometer (model 680; Bio-Rad Laboratories, Hercules, CA). In order to get insight into cell death we performed confocal microscopy. The cells were exposed to Co/TiO2 nanofibers for specific time duration as abovementioned. In order to distinguish live cells from dead cells, the exposed cultures were stain with LIVE/DEADR Cell-Mediated Cytotoxicity Kit (L7010) contents. The images were acquired on an Inverted Axiovert 200 M BP CLSM equipped with laser and scanning module in an epifluorescence mode. Images were saved using Software LSM (Zeiss image confocal software) and exported to Adobe PhotoShop for digital processing. Further to check the cell attachment on nanofibrous matrix, chemical fixation of cells was carried out in each sample. The samples were rinsed twice with phosphate buffered saline (PBS) and subsequently fixed with 2.5 % glutaraldehyde. After cell fixation, samples were rinsed with distilled water to remove the remaining glutaraldehyde. Furthermore, the cells attached to nanomatrix were serially dehydrated with graded ethanol concentrations (20–100 % ethanol). Finally the morphology was observed by SEM.
3 Results and discussion
In summary, we evaluated the effects of Co-doped TiO2 nanofibers on the morphology and cytotoxicity of C2C12 cells. Previously Karlsson et al.  has demonstrated the size dependent toxicity of metal oxide particles. On the other hand a number of studies have demonstrated the benefits of pristine titanium dioxide and its composites. For instance; Eun et al.  demonstrated the cytocompatibility of SiO2–TiO2 composites whereas Groenke et al.  studied the potential health and environmental effects of the nano-TiO2. Recently; cobalt based composites are being used for biomedical purposes such as cobalt-containing titanium phosphate-based glass for engineering of vascularized hard tissues . However; there is limited information about the possible impact of Co-doped TiO2 nanofibers on human health or the environment. To our knowledge, this is the first study in which C2C12 mouse myoblasts were used to evaluate the beneficial/toxicological effects of Co-doped TiO2 on cells. From the in vitro test results, it was concluded that Co-doped TiO2 nanofibers could support growth of myoblasts, however the cell compatibility decreases with high doses and after sustained exposure. It was also observed that low concentration of Co-doped TiO2 naofibers showed a beneficial effect on the growth of myoblast cells. Thus suitable modifications of this material and detailed understanding in this area are expected to lead to considerable developments in biomedical sciences and hence the Co-doped TiO2 nanofibers would be successfully exploited for various tissue engineering applications. On the basis of present observations we can conclude that cobalt based titanium dioxide composite nanofibers could be employed as extracellular matrix for C2C12 adhesion and growth. However, in light of the decreased biocompatibility observed with increasing Co concentration, detailed biocompatibility testing and subsequent biofunctionalisation are required to realize the potential of Co-doped materials (such as Co-containing phosphate glasses and Co based alloys etc.) for use in tissue engineering and muscle cell growth.
Although some information about the interaction between C2C12 cells and nanotextured materials exist, however, the cytotoxicity in biological systems exposed to nanomaterials is often inconsistent and even contradictory. The present work was aimed to explore the interactions between the C2C12 cell line and cobalt doped titanium dioxide nanofibers. To sum up; novel Co-doped TiO2 nanofibers were prepared via electrospinning process. The morphology of the resulting composite nanofibers was analyzed by SEM and TEM and the attachment of C2C12 cells on nanofibers was analyzed by Bio-SEM respectively. The crystallinity of the synthesized nanofibers was described by XRD whereas the elemental composition was determined by EPMA and EDX spectroscopy. In order to investigate the strength of the electrospun Co-doped TiO2 matrix for cell proliferation and to check their in vitro cytocompatibility, a standard CCK-8 assay was used. From the in vitro test results, it was concluded that low Co-doped TiO2 nanofibers content proved beneficial for the adhesion and propagation of myoblast cells; however the cell compatibility decreases with high doses and after sustained exposure. Collectively, these results demonstrated reasonable biocompatibility of the Co-doped TiO2 at low concentration in in vitro assays. However, for practical use, a detailed study is required to check the toxicity behavior and other physicochemical aspects in vivo model.
This work was fully supported by research funds of Chonbuk National University in 2013. This work has been partly supported by a grant from Next-Generation Bio-Green 21 Program (Project Nos. PJ008191 and PJ008196) Rural Development Administration, Republic of Korea. Dr. Touseef Amna sincerely acknowledges financial support from Research Academic Promotion Programme of Chonbuk National University.