- Nano Express
- Open Access
PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration
© Haider et al.; licensee Springer. 2014
- Received: 16 April 2014
- Accepted: 19 June 2014
- Published: 25 June 2014
The development of tissue engineering in the field of orthopedic surgery is booming. Two fields of research in particular have emerged: approaches for tailoring the surface properties of implantable materials with osteoinductive factors as well as evaluation of the response of osteogenic cells to these fabricated implanted materials (hybrid material). In the present study, we chemically grafted insulin onto the surface of hydroxyapatite nanorods (nHA). The insulin-grafted nHAs (nHA-I) were dispersed into poly(lactide-co-glycolide) (PLGA) polymer solution, which was electrospun to prepare PLGA/nHA-I composite nanofiber scaffolds. The morphology of the electrospun nanofiber scaffolds was assessed by field emission scanning electron microscopy (FESEM). After extensive characterization of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectrometry (EDS), and transmission electron microscopy (TEM), the PLGA/nHA-I and PLGA/nHA (used as control) composite nanofiber scaffolds were subjected to cell studies. The results obtained from cell adhesion, alizarin red staining, and Von Kossa assay suggested that the PLGA/nHA-I composite nanofiber scaffold has enhanced osteoblastic cell growth, as more cells were proliferated and differentiated. The fact that insulin enhanced osteoblastic cell proliferation will open new possibilities for the development of artificial scaffolds for bone tissue regeneration.
Polymeric fibers have been fabricated using various techniques such as self-assembly, phase separation, melt spinning, and electrospinning. Among these, electrospinning is a unique, simple, cost-effective, versatile, and scalable technique used for the fabrication of nanofibers from a wide range of natural and synthetic polymers [1–4]. Electrospinning is used frequently in the engineering, environmental, and biomedical fields [5, 6]. Fibrous scaffolds prepared via electrospinning exhibit unique properties such as a high surface area-to-volume ratio, ultrafine uniform fibers, having high porosity and variable pore size distribution within the intra-fibrous structure . These properties serve to enhance the biocompatibility and biological responses of the scaffold. The electrospinning process is quite flexible; therefore, it provides more efficient control over the nanofiber scaffold by altering several governing experimental parameters, including polymer concentration, voltage, and needle to collector distance . Until now, a variety of synthetic as well as natural biopolymers have been used to date for the preparation of fibrous scaffolds by electrospinning [8, 9]. Among synthetic polymers, poly(lactide-co-glycolide) (PLGA), a biodegradable polyester, has been studied extensively in the preparation of electrospun scaffolds. Apart from biocompatibility, PLGA exhibits excellent biodegradability over time and its degradation rate can be altered by adjusting the monomer ratio [10, 11]. A series of experiments have concluded favorable cellular responses to these nanofibrous scaffolds; Kim et al. demonstrated enhanced osteoblast adhesion and proliferation onto electrospun nanofiber scaffolds .
Inorganic nanomaterials such as nanotubes, nanocrystals, nanorods, nanospheres, nanoparticles, and nanofibers have unique properties, which cannot be achieved by using pristine polymers. During the electrospinning process, several inorganic fillers, including β-tricalcium phosphate (β-TCP), hydroxyapatite nanorods (nHA), multiwall carbon nanotubes (MWCNT), and calcium carbonate (n-CaCO3) are successfully incorporated into the polymer solution to fabricate biocomposite electrospun scaffolds for tissue engineering . HA is among one of the widely used bioceramic material having similar composition and morphology to the inorganic component of natural bone . In addition, it can provide a favorable environment for cell adhesion, osteoconduction, and osteoinduction.
Controlling the surface energies enables us to precisely control the surface and interfacial properties of nanomaterials ranging from wetting to adhesion, thus providing an active site for chemical reactions and/or interactions with foreign bodies. This can be achieved by tailoring the surface of nanomaterials [2, 13]. Recently, several reports have described strategies for surface modification, including the chemical attachment of long or short-chain molecules to a wide range of surfaces or substrates [14, 15]. Succinic acid is used as a surface modifier and carrier for targeted drug delivery systems (DDS) on nanomaterial surfaces due to its non-immunogenic, non-toxic, and non-antigenic properties . Succinic acid can alter the physical and chemical properties of the substrates , where the substrate surfaces modified by succinic acid are more prone to chemical reactions with suitable functional groups such as the primary amine group (NH2). The functional groups provide active sites for the covalent conjugation of the protein with other macro- and micromolecules and hence improve the biocompatibility and dispersion properties of the substrate. The incorporation of bioactive agents by mixing, encapsulation, or covalent bonding to electrospun fibers could lead to advanced biofunctional tissue engineering (TE) scaffolds . The biofunctionalization of electrospun fibers is, however, the most prominent method used and determines the efficiency of these fibers to regenerate biofunctional tissues. Insulin is a peptide protein capable of regulating carbohydrate and fat metabolism in the body . It is highly effective in controlling diabetes mellitus and is used in the treatment of diabetes . In addition, insulin is a well-known cell growth factor capable of enhancing cell proliferation, including activation of muscle stem cells [20–22]. Therefore, several insulin-like growth factors were used previously in the field of bone regeneration, which showed high biocompatibility and enhanced cell growth .
The aim of the present study was to enhance the cell affinity, osteoconduction, and osteoinduction by grafting insulin onto the surface of nHA by chemical reaction, which was used to fabricate three-dimensional electrospun PLGA/nHA-I composite nanofiber scaffolds. The adhesion, proliferation, and differentiation of MC3T3 cells were investigated to evaluate the potential of the PLGA/insulin-grafted nHAs (nHA-I) nanofiber composite as a bone TE scaffold.
PLGA (lactide/glycolide 85:15), with molecular weight of 240,000, insulin from the human pancreas, and succinic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). nHA was synthesized in the laboratory. Minimal essential medium (MEM)-alpha and the osteoblast MC3T3-E1 cell line were purchased from the Korea cell bank (Seoul, South Korea). 5-Bromo-2-deoxyuridine (Brdu) and alizarin red staining kits were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA) and Millipore (Billerica, MA, USA), respectively. Fetal bovine serum (FBS) and penicillin G-streptomycin were purchased from Gibco, Tokyo, Japan. All reagents and chemicals in this study were used without any further purification.
Synthesis of nHA
nHA was synthesized via chemical precipitation, as previously described . Briefly, 400 ml (NH4)2PO3 and 300 ml CaNO3 · 4H2O solutions were prepared separately by dissolving 19.75 g (NH4)2PO3 and 57.5 g (CaNO3) · 4H2O in distilled water. The pH of (CaNO3) · 4H2O solution was adjusted to 10.4 with NH4OH, after which the two solutions were mixed dropwise with vigorous stirring. During mixing, a white precipitate was formed, which was aged for 4 days to form nHA. The synthesized nHA was washed with distilled water until the pH reached 7. The nHA was resuspended in 1-butanol to prevent nHA from aggregation during the drying process. Finally, the precipitate was dried at 80°C and calcined at 500°C for 4 h to remove rudimental organic compounds.
Surface grafting of nHA via insulin
Solution preparation and electrospinning
PLGA polymer solution in the concentration range of 5 to 20 wt.%, was prepared by dissolving in a binary solvent (THF and DMF in a 3:1 ratio). The solution was stirred overnight at room temperature until complete dissolution. The solution was then subjected to electrospinning. For this, the PLGA solution was placed into a 10-mL glass syringe fitted with a needle of 0.9 mm (20 G) inner diameter. A typical electrospinning setup consists of four main components: (i) a pump, to hold and pump the hypodermic syringe containing polymer solution, which allowed controlled outflow of the polymer solution; (ii) a high voltage supply of 1 to 50 kV; (iii) a metallic capillary (needle) connecting the syringe to the positive voltage; and (iv) a metallic collector (flat or rotating drum), which can either be stationary or rotating) connected to negative voltage. The electrospinning process began when a high electric current was generated from the power supply. The solution moved to the tip of the needle, and the hemispherical shape of the droplet was destabilized by charges that accumulated on its surface. As the charges balanced the fluid surface tension of the polymer solution, the droplet was converted to a Taylor's cone with a semivertical angle of approximately 30° . At a critical electrical voltage, the electric forces surpassed the surface tension of the droplet and a jet of ultrafine fibers emanated from the tip of the Taylor's cone and was collected onto the collector kept at fixed distance . Due to the high electric voltage used in the process, the fluid jet usually remains stable for a small distance (2 to 4 cm) before scattering. The optimized electrospinning conditions used in the present study were tip-to-collector distance 20 cm, applied voltage 20 kV, needle diameter 20 G (0.9 mm), and flow rate 1 mL/h. The electrospun nanofibers collected were removed from the collector and dried overnight at 40°C to remove the remaining solvent. After drying, the sample was sputter-coated with gold and its morphology was observed by field emission scanning electron microscopy (FESEM; 400 Hitachi, Tokyo, Japan). The same procedure was adapted for the preparation of the electrospun PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds. Briefly, both pristine nHA and insulin-grafted nHA-I were added into the PLGA polymer solution and were mechanically dispersed via alternate stirring and sonication. After dispersion, the samples were subjected to electrospinning process.
Osteoblastic cell culture
To examine the interaction of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds with osteoblastic cells (MC3T3-E1), the composite nanofiber scaffolds were cut into small circular discs, fitted inside a 4-well culture dish, and immersed in MEM medium containing 10% FBS (Gibco; Invitrogen, Carlsbad, CA, USA). Subsequently, 1 mL of the MC3T3-E1 cell solution (3 × 104 cells/mL) was added to the surface of the composite nanofiber scaffolds and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 1 and 3 days. After incubation, the supernatant was removed and the composite nanofiber scaffolds were washed twice with phosphate-buffered saline (PBS; Gibco, Langley, OK, USA) and fixed in a 2.5% glutaraldehyde solution for 15 min. The samples were then dehydrated, dried in a critical point drier, and sputter-coated with gold. The surface morphology of the composite nanofiber scaffolds was observed by FESEM (400 Hitachi; Tokyo, Japan).
To evaluate the cytoskeletal organization of cells onto the PLGA/nHA-I and PLGA/nHA composite as well as pristine PLGA nanofiber scaffolds, double staining was performed according to the manufacturer's protocol. Briefly, osteoblast cells were seeded onto the scaffolds (2 × 104 cells/mL) and were cultured for 3 days. The cells were fixed with 4% paraformaldehyde in PBS. After fixation, the samples were washed using PBS buffer solution containing (0.05% Tween-20). The samples were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 25°C and then incubated for 30 min in PBS containing 1% bovine serum albumin (BSA). This was followed by the addition of 5(6)-tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (Millipore) (TRITC) for approximately 1 h. The samples were washed three times (10 min each) using the buffer solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Millipore) for 5 min. Fluorescence images were visualized using a confocal laser scanning microscope (model 700; Carl Zeiss, Oberkochen, Germany) after washing the scaffolds three times (10 min each) with the buffer solution.
Proliferation of MC3T3 osteoblastic cells seeded on the PLGA/nHA-I, PLGA/nHA composite, and pristine PLGA nanofiber scaffolds was determined using a colorimetric immune assay, based on the measurement of BrdU, which was incorporated during DNA synthesis. BrdU enzyme-linked immunosorbent assay (ELISA; Roche Molecular Biochemicals) was performed according to the manufacturer's instructions. Briefly, after cell culture for 48 h, BrdU-labeling solution was added to each well. The solution was allowed to incorporate into the cells in a CO2 incubator at 37°C for 20 h. Subsequently, the supernatant in each well was removed by pipetting and washed twice with PBS. The cells were treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, Tokyo, Japan) and harvested by centrifugation of the cell solution at 1,000 rpm for 15 min. The harvested cells were mixed with FixDenat solution to fix the cells and denature the DNA and then incubated for 30 min. Subsequently, diluted anti-BrdU peroxidase (dilution ratio of 1:100) was added to the cells and incubated at 20°C for 120 min. After removing the unbound antibody conjugate, 100 μL substrate was added and allowed to stand for 20 min. The reaction was completed by adding 25 μL H2SO4 solution (1 M). The solution was then transferred to a 96-well plate and measured within 5 min at 450 nm with a reference wavelength of 690 nm, using an ELISA plate reader (EL 9800). The blank reading corresponded to 100 μL of culture medium with or without BrdU.
Alizarin red staining
Alizarin red staining of the MC3T3 osteoblastic cells cultured on the electrospun PLGA/nHA-I, PLGA/nHA, and pristine PLGA nanofiber scaffolds was performed to examine mineralization and differentiation. Briefly, after culturing the MC3T3 osteoblasts, the medium was aspirated without disturbing the cells. The culture dish with the osteoblastic cells was washed twice with PBS. The cells were then fixed with 10% formaldehyde and incubated for 15 min at room temperature. The fixative reagent was removed carefully, and the cells were rinsed three times (10 min each) with distilled water to avoid disturbing the monolayer. After washing, the excess water was removed and alizarin red staining solution (1 mL/well) was added to the cells and the samples were incubated for 30 min. Subsequently, the excess amount of dye was removed from the stained cells by washing the samples four times with distilled water (5 min each) with gentle rocking. Digital images of the stained cells were obtained with a camera (Nikon E 4500, Tokyo, Japan).
Von Kossa assay
Calcium deposition of MC3T3-E1 cells was examined by Von Kossa staining. The cells were cultured for 15 days on PLGA/nHA-I, PLGA/nHA, and pristine nanofiber scaffolds under the same conditions as those described in the alizarin red staining experiment. After incubation, the cells were washed three times with PBS for 5 min, fixed with 10% formaldehyde for 30 min, and washed three times with distilled water for 10 min. The fixed samples were treated with 5% AgNO3 solution for 5 min under ultraviolet radiation. After removing the AgNO3 solution, the samples were washed with PBS twice followed by the addition of 5% Na2S2O3 solution to the plate and allowing the plates to stand for 5 min. Finally, the samples were washed twice with distilled water and digital images of the stained cells were obtained.
The results are displayed as the mean ± standard deviation. The statistical differences were determined using a student's two-tailed test. Scheffe's method was used for the multiple comparison tests at a level of 95%.
Preparation of nanofiber scaffolds
Fourier transform infrared spectroscopy study
X-ray photoelectron spectroscopy analysis
Chemical composition of nanofiber scaffolds calculated from ESCA (XPS) survey scan spectra
Atomic weight (%)
X-ray diffraction spectroscopy study
Transmission electron microscopy (TEM) morphology study
Bioactivity and cellular response
The adhesion behavior of the osteoblastic cells to implantable materials is determined mostly by their surface chemistry and topography . To elucidate the in vitro osteoblastic cell behavior and assess the effectiveness of insulin grafting onto the surface of nHA, osteoblastic cells were cultured on pristine PLGA nanofiber scaffolds as well as PLGA/nHA and PLGA/nHA-I composite nanofiber scaffolds. As depicted in Figure 7, more cells adhered to the PLGA/nHA-I composite nanofiber scaffolds (Figure 7c,f) contrary to the PLGA/nHA composite (Figure 7b,e) and pristine PLGA nanofiber scaffolds (Figure 7a,d). The increased adhesion of osteoblastic cells to PLGA/nHA-I composite nanofiber scaffolds was attributed to the presence of nHA-I in the PLGA nanofiber scaffold (PLGA/nHA-I) and to the rough morphology of the PLGA/nHA-I composite nanofiber scaffolds due to the protrusion of the nHA-I from the PLGA nanofiber scaffolds (Figure 6d). Insulin has the capability of enhancing cell growth [20, 22], whereas protrusion makes the surface of the scaffold rough. Osteoblastic cells adhesion was enhanced in both cases [20, 22, 34, 36]. The order of increase in cell adhesion and spreading of osteoblastic cells was PLGA/nHA-I > PLGA/nHA > PLGA. Besides the type of scaffolds, adhesion of the osteoblastic cells was also increased with an increase in incubation time from 1 to 3 days. In addition to better adhesion, more spreading of osteoblastic cells was observed on the PLGA/nHA-I composite nanofiber scaffold as compared to the PLGA/nHA composite and pristine PLGA nanofiber scaffolds.
Alizarin red staining
Von Kossa assay
Insulin was grafted on the surface of hydroxyapatite nanorods to produce surface-modified (nHA-I) composite nanofiber scaffolds, composed of PLGA and nHA-I obtained by blending of nHA-I with PLGA and subsequent electrospinning. After confirming the presence of nHA-I in the PLGA matrix, the scaffolds were subjected to the cell culture studies for assessing their biocompatibility and bioactivity. The results obtained from the in vitro studies indicate that the cell adhesion, proliferation, and differentiation of the osteoblastic cells were accelerated on PLGA/nHA-I composite nanofiber scaffold as compared to PLGA/nHA composite and pristine PLGA nanofiber scaffolds. This study will prove a potential step forward in triggering research on bone tissue engineering, bone remodeling, artificial bone implantation, and site-specific drug delivery for various bone diseases.
This work was supported by the general research program (2013.RIA 2005148) from the Ministry of Education, Science and Technology of South Korea, and the Basic Research Laboratory program (no. 2011-0020264).
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