Enhancement of Fibroblasts Outgrowth onto Polycaprolactone Nanofibrous Grafted by Laminin Protein Using Carbon Dioxide Plasma Treatment
Mohammad Ali Sahebalzamani 1*, Mohammad Taghi Khorasani 2, Morteza Daliri Joupari 3
1 Department of Biomaterials, Faculty of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2 Department of Polymeric Biomaterial, Iran Polymer and Petrochemical Institute, Tehran, Iran.
3 Department of Animal and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Received: Jun. 10, 2017; Accepted: Aug. 14, 2017; Published: Sep. 7, 2017
Citation: Mohammad Ali Sahebalzamani, Mohammad Taghi Khorasani, and Morteza Daliri Joupari, Enhancement of Fibroblasts Outgrowth onto Polycaprolactone Nanofibrous Grafted by Laminin Protein Using Carbon Dioxide Plasma Treatment. Nano Biomed. Eng., 2017, 9(3): 191-198.
A common approach in tissue engineering is to mimic the architecture of the natural extracellular matrix (ECM). The ECM plays an important role in regulating cellular behaviors by influencing cells with biochemical signals and topographical cues. Nanofibrous constructs have been used extensively as potential tissue engineering platforms. It is generally hypothesized that a close imitation of the ECM will provide a more conducive environment for cellular functions ranging from adhesion, migration, proliferation to differentiation. In this study, the polycaprolactone (PCL) nanofibers designed were then modified by carbon dioxide plasma and laminin in order to enhance the cell adhesion, spreading and proliferation. The samples were evaluated by attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscope (SEM), contact angle and finally, cell culture. ATR-FTIR structural analysis showed the presence of functional groups on the nanofibrous surfaces. The SEM images showed the average diameter of nanofibers to be about 100 - 300 nm for samples. The 82° difference was obtained in the contact angle analysis, obtained for the laminin-modified nanofibrous mat against the unmodified nanofibrous mat. Cellular investigation showed better adhesion and cell growth and proliferation of laminin-modified nanofibrous samples than other samples. Therefore, the modification of electrospun scaffolds with bioactive protein is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion and proliferation.
Keywords: Porous nanofibrous polycaprolactone; Physical adsorption; Carbon dioxide plasma treatment; Enhance the cell adhesion
Tissue engineering is a rapidly growing area that aims to create, repair or replace tissues and organs by using combinations of cells, biomaterials, and biologically active molecules. The biomaterial scaffold plays a pivotal role in most tissue engineering strategies. To guide the organization, growth and differentiation of cells in tissue engineered constructs, the biomaterial scaffold should be able to provide not only a physical support for the cells but also the chemical and biological cues needed in forming functional issues . Polycaprolactone (PCL) is a biomaterial that is used in a variety of applications including surgical sutures, wound dressing, drug delivery and tissue engineering. This is due to its specific properties such as good biocompatibility, biodegradability, non-toxicity as well as appropriate mechanical strength. However, this material is a hydrophobic polyester which should be modified with other materials until it improves its cell adhesion and hydrophilicity properties [2, 3]. It has been scientifically proved the extracellular matrix (ECM) mimics improve the attachment, proliferation and the viability of the cultured cells. Electro-spinning has been recently developed into a technique to prepare nanofibers with the diameter ranging from tens of nanometers to several microns . The electro-spun fibrous mats also show extremely high surface area and large porosity. Besides, the fibrous structure of the electro-spun mats mimics the extracellular matrix [5, 6]. Some natural materials such as collagen, fibronectin and laminin have been reported as scaffold modifiers. Controlling surface properties is so important for the high performance of adhesion . Biomaterials wettability is a necessary factor in the surface modification of materials. Also, surface modification is an integral element for better growth and proliferation of cells on the nanofiber scaffold. Plasma-surface modification (PSM) is an effective and economical surface treatment technique for many materials and of growing interests in biomedical engineering. Non-thermal and low-pressure plasma have been used in a series of surface treatment applications. The majority of plasma-assisted technologies are based on low pressure processes . The treatment of polymeric materials with plasma is a frequently used technique to accomplish surface modifications that affects chemical composition as well as surface topography . Moreover, microwave discharges are routinely employed in the processing of materials to deposit films as well as coatings . Laminin is a component of the basement membrane and contains the RGD sequence (contains the three-amino-acid sequence arginine-glycine-aspartic acid binding sequence that reacts with integrin receptors on the growth cone and promotes cellular adhesion) that aids in cell adhesion and the five aminoacid sequence isoleucine-lysine-valine-alanine-valine sequence that controls neurite outgrowth . Ko et al. used laminin as the contact guidance biochemical cues for axonal outgrowth . Laminin is one of the ECM component that is continuously synthesized after nerve injury and it plays a crucial role in cell migration, differentiation and axonal growth . For example, myelination in the peripheral nervous system (PNS) is affected by laminin. Studies have described that even as Schwann cells could proliferate and migrate along axons, differentiation of myelinating phenotype was not observed without the presence of laminin . Furthermore, in-vitro experiments have shown that neurite outgrowth is enhanced on scaffolds that were covalently bound with laminin . These studies showed that in-vitro directional guidance of the neurite outgrowth was achieved and enhanced using scaffolds that were physically adsorbed with laminin. Improved axonal outgrowth has also been observed in nerve guides filled with laminin gel as well. Therefore, the incorporation of laminin onto nanofibers can potentially improve the rate of nerve regeneration [16-18]. In this research, the modified PCL nanofibers were obtained through the plasma radiation method with carbon dioxide gas. The samples were evaluated by attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscope (SEM), water contact angle and also the cell culture with fibroblasts cells.
Materials and Methods
PCL with 80,000 (g/mol) molecular weight was purchased from Sigma-Aldrich (St. Louis, MO), and hexafluoro-2-propanol (HFP), the solvent system for PCL was purchased from Sigma-Aldrich and used as received without further purification. Electrospinning apparatus used in this study was prepared from Asia nano meghyas company (Iran). PCL was dissolved at determined concentration in HFP. The PCL solution (9% w/v) was contained in a glass syringe controlled by syringe pump. A positive high voltage source through a wire was applied at the tip of a syringe needle. In this situation, a strong electric field was generated between PCL solution and a collector. When the electric field reached a critical value with increasing voltage, mutual charge repulsion overcame the surface tension of the polymer solution and an electrically charged jet was ejected from the tip of a conical shape as the Taylor cone. The solution was electrospun from a 10 mL syringe with a needle diameter of 15 mm and mass flow rate of 1 mL/h. A high voltage (20 kV) was applied to the tip of the needle attached to the syringe when a fluid jet was ejected. The linear rate of the rotating disk (drum) was set to 1,000 rpm. The resulting fibers were collected on 15-mm cover slips placed on respective collectors. The used parameters for this nanofibers preparation can be seen in Table 1. Ultra-fine fibers were formed by narrowing the ejected jet fluid as it underwent increasing surface charge density due to evaporation of the solvent. An electrospun PCL nanofibrous mat was carefully detached from the collector and dried in vacuum for 2 days at room temperature to remove solvent molecules completely.
Fig. 1 Schematic of modification with laminin by carbon dioxide plasma treatment and physical adsorption.
Table 1 Parameters for nano fibers preparation