Design and development of two novel 3D conductive thread scaffolds for nerve tissue engineering
Creators: name: Sadeghi Taheri, Nooshin
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320606
subject: Regenerative medicine (incl. stem cells)
classificationCode: 320902
subject: Cellular nervous system
classificationCode: 400302
subject: Biomaterials

description: Millions of patients suffer from peripheral nerve injuries developed as a result of damage to peripheral nervous system. Nerve tissue engineering is a promising approach to repair damaged or malfunctioning peripheral nerves (auto graft) caused by accident or disease. There are different approaches to achieve this; one is to combine a fabricated scaffold made from one or two materials with appropriate properties with specific cells and physical cues to motivate cells to develop into functional tissues. Different kind of tissues has specific challenges and requirements, from the shape of the scaffold (e.g., tubes, sponges, blocks etc) and the physical properties of the biomaterials (e.g., porosity, degradability, stiffness) to the ultimate biological results (e.g., promoting cell proliferation, differentiation, cell invasion).<br><br>The field of tissue engineering and biomaterial development demand a range of expertise, since all tissue engineering areas require different materials and fabrication strategies and planning. The form of implants varies between the applications. Some applications such as muscle or fat need the replacement of large blocks of tissues. Others like replacing the skin in healing wound need a membrane. To engineer an implant for nerve or muscle fibers, fibrous structures are considered. After adapting material and scaffold properties to different tissues in the body, the most important property essential for all tissue engineering is biocompatibility. This property means that the material and scaffold does not have toxicity and will not induce any harmful effect towards considered tissues or cells. In this study, two 2D materials (GO, MoS2) with excellent properties are introduced to be combined with common cotton thread as scaffold for the first time in nerve tissue engineering.<br><br>The aim of this project is to discover an alternate substrate like thread and other materials that enhance conductivity, permeability and better electrical guidance for nerve tissue scaffolds. Current tissue scaffold system has limited permeability due to the lack of the vascular system. The use of cotton thread can be used to mimic the vascular system to promote permeability, in terms of moving nutrients, removing waste and other biomolecules. Conductive materials allow electrical stimulation which may be advantageous for nerve tissue regeneration and to control electrical signal direction through wettability of the thread. Chapter 3 aims to investigate the biocompatibility and conductivity of the reduced graphene oxide coated threads as a tissue engineering scaffold. To achieve this, first graphene oxide was synthesized followed by coating the CPAM treated threads with graphene oxide particles. Next graphene oxide was reduced by two method of reduction; electrochemical reduction and metal iodide treatment. The resulting scaffolds exhibited proper conductivity in both methods and concentrations of graphene oxide, however, electrochemical reduction of GO proved better conductivity compared to metal iodide treatment method. As far as permeability is concerned, electrochemical reduction exhibited improved wicking properties of thread scaffolds, which may play a role in better neurite exchange and guiding electrical activity along the thread scaffolds within the cell culture system. The last step was to investigate biocompatibility of rGO coated thread, with a promising outcome confirming that rGO-thread scaffolds are biocompatible. Overall, this study showed that rGO thread scaffold can be a promising nerve tissue scaffold for nerve tissue engineering applications.<br><br>The conception of the second project is initiated in emergent of molybdenum disulphide (MoS2) as its recent progress suggest its new interesting applications in biomedical field and existing graphene with its excellent properties to be investigated and compared for better tissue scaffolds materials. In Chapter 4, molybdenum disulphide was synthesized and coated to the treated cotton thread. Molybdenum disulphide was successfully rendered conductive by electrochemical intercalation and characterized by XPS, RAMAN and SEM. The wicking properties of rMoS2-thread were determined. And finally, rMoS2-thread showed biocompatibility to nerve cells.<br><br>These developed tissue scaffolds were surface functionalized by CDI treatment conjugated with gelatin to enhance cell adhesion on the tissue scaffolds.<br><br>The three-dimensional thread scaffolds were assessed for permeability, cell adhesion and biocompatibility in Chapter 5. First approach was to enhance and promote attachment of nerve cells (NG108-15) seeded onto the coated thread scaffolds to the surface of tissue scaffold. Surfaces and layers of coated (rGO 2%, rMoS2) thread scaffolds were engineered by treatment of CDI conjugated with gelatin solutions to enhance neural cell adhesion. Before cell seeding onto the surface functionalized thread scaffolds, the accessible amine content on the surface of all samples were investigated using Acid orange II assay. In both tissue scaffolds (rGO2%, rMoS2) amine content was successfully increased by surface functionalization to promote cell adhesion. By developing a new protocol to optimize CDI/Gelatin tissue scaffold’s surface functionalization cell attachment was improved in both tissue scaffolds. Then cell adhesion was investigated by Immunofluorescence assay under confocal laser microscopy. Second approach was to investigate permeability of coated thread scaffolds after surface functionalization. The permeability of these three dimensional tissue scaffolds was determined by wettability assay. The wettability assay revealed a decrease of permeability in both tissue scaffolds after surface functionalization. Plasma treatment was carried out on all thread samples to facilitated liquid wicking properties of the surface functionalized (rGO2%, rMoS2) tissue scaffolds in two folds.<br><br>Electrical stimulation of NG108-15 cells attached on the thread scaffolds was established and investigated in Chapter 6 for proliferation and cell functionality. The proliferation rates of the electrically stimulated nerve cells were significantly higher than the non-stimulated ones. Excitability of neurons in engineered nerve tissue scaffolds were determined by optical imaging of cell signaling using voltage-sensitive dyes. These findings highlight for the first time the possibility of enhancing nerve regeneration through conductive thread scaffolds with excellent surface properties and cell proliferation. Our novel integration of engineered cotton thread with reduced graphene oxide (rGO2%) and new biomaterial rMoS2 as a 3D nerve tissue scaffold provides a favourable nerve conduit for peripheral nerve repair and other neural applications. Third approach was to establish electrical stimulation properties for NG108-15 cell line by investigating new electrical stimulation setting, voltage, frequency and duration to properly excite NG108-15 neural cells. A potential of 60 mV/mm for 5 min at 5 Hz was selected with best results to be applied to the cells through customized electrical 6-well plate system using a signal generator. To carry out the electrical stimulation, two platinum plates attached to the customise 6-well plate lid, were in contact with the cells though medium. Direct current was applied to the cells to regulate cell functions. Fourth approach is to electrically stimulate neural cells NG108-15 while seeded and attached to the conductive thread tissue scaffolds. After electrical stimulation of the cell-scaffolds, it is required to investigate cell viability, proliferation and functionality of scaffolds. Cell-scaffolds viability was investigated by alamarBlue® assay, and then proliferation rates of the electrically stimulated cell-scaffolds were carried out by PicoGreen® assay, with significantly higher rate than non-stimulated cell-scaffolds. Then for the first time, excitability of our new developed engineered nerve tissue scaffolds was determined and recorded by optical imaging of cell signaling using voltage-sensitive dyes (FluoVolt™ membrane assay kit).<br><br>To the best of author’s knowledge, there are no prior reports on employing conductive common cotton thread in nerve tissue engineering and nerve repair. This is the first time that reduced graphene oxide coated thread has been used as a nerve tissue scaffolds. Also, employing molybdenum disulphide to make cotton thread conductive to be used as a nerve tissue scaffold is a novel idea to create a second nerve tissue scaffold for nerve tissue repair.