Functionalized nanofiber based scaffold in nerve tissue engineering

Spinal cord injury (SCI) is a destructive disorder which leads to chronic pain and permanent disabilities due to motor and sensory dysfunctions. The major causes of SCI are sports and traffic accidents, falls and gunshots. According to statistical data of National Spinal Cord Injury Statistical Center, 13.400 new cases occur each year in US and as the spinal cord have very limited capacity to regenerate, the injury becomes permanent. There are some supportive treatments such as rehabilitation and physical therapy. Besides, there are some ongoing clinical trials. However, complete solution of SCI has not been discovered yet. Tissue engineering approaches are used in order to restore and regenerate the damaged tissues and cells. Therefore, many researchers are focusing on tissue engineering strategies for treatment of spinal cord injuries in order to regenerate the neurons and re-connect the transected neural circuits. Main idea in tissue engineering is to support the injury site with a scaffold which can mimic the 3-D structure of native environment. Autogenic, allogeneic or xenogeneic cells can also be transplanted together with the scaffolds in order to replace the injured cells and enhance regeneration by the secretion of growth factors and other ECM proteins which can trigger the host cells to proceed repair. In neural tissue engineering, there are different types of scaffolds constructed, but the most promising results have been achieved from micro and nanofiber structures as they have similar morphologies to nerves. Various cell types such as embryonic stem cells, mesenchymal stem cells, Schwann cells have also been used in many studies for treatment of SCI. However, researches are still going on to obtain optimum scaffold. To construct neural tissue engineered scaffolds, different biomaterials can be used and biodegradable aliphatic polyesters are one of the most preferred polymers. PLLA is a biodegradable thermoplastic aliphatic polyester and it is frequently used in tissue engineering applications. PBSu is another biodegradable thermoplastic aliphatic polyester which has been extremely used in industrial applications. However, biomedical applications of PBSu is very limited and it is a rather novel polymer for biomedical applications. Especially PBSu has not been studied in neural tissue engineering studies earlier. The aim of this study is to investigate an optimal scaffold structure from aligned and functionalized nanofibers and supportive cells which can provide regeneration of the mechanically disrupted neurons and axons in SCI. In order to achieve our goal, above mentioned strategies were combined and an integrated scaffold structure was proposed in order to be used to treat transected nerves. The two main parts of the design are the outer sheath and the inner matrix. We suggested to build a porous cylindrical structure for the outer sheath of our designwhich provides the integrity of the construct and the inner matrix was designed to be constructed from aligned nanofibers which mimic the native neural tissue and reconnect the neural circuit. These carrier part of the scaffold was prepared from PLLA polymer blended with PBSu polymer. One of the significant characteristics of the design is the modification of the surface of the aligned nanofibers with synthetic peptides. The nanofibers are functionalized by neuro-active peptide YIGSR which is derived from Laminin-I protein in order to trigger the differentiation of the cells in to mature neurons and support regeneration in the injured site. Especially, it is expected that the MSCs seeded scaffolds can fasten the healing process, provide axon extensions and stem cell differentiation. In this study, the effects of the chemically bound peptides on aligned nanofibers were investigated. In order to increase the regeneration potential, Mesenchymal Stem cells and Schwann cells were also combined with the proposed scaffold. MSCs were seeded on the nanofibers which were located in the cylinder and these cells have the capacity to differentiate into mature neurons and have the potential to restore the injured tissue. In addition to MSCs, the inside walls of the outer cylinder were seeded with the Schwann cells which are expected to secrete neural growth factors to enhance regeneration and provide differentiation of MSCs. In this study, we first built the outer sheath of the design from PLLA/PBSu polymer blend. Different blend ratios were chosen and polymeric sponges were constructed via solvent casting and particulate leaching method. The constructed sponges were visualized via Scanning Electron Microscope and the porosity of the surfaces were analyzed. In vitro degradation profiles of the scaffolds were also investigated by 120 days of incubation. In order to evaluate the biocompatibility of the different blend ratios, Schwann cells were cultivated on polymeric sponges and in vitro cell culture studies were performed. According to the results of these studies, the optimal blend ratio was determined as PLLA/PBSu (3%, w/v, 2:1). For the inner matrix, aligned nanofibers were constructed via electrospinning. There are very limited studies considering PBSu nanofiber fabrication, especially for tissue engineering purposes. Therefore, many different solvents and polymeric ratios were investigated. PBSu was blended with PLLA for aligned nanofiber construction and the scaffolds were visualized via Scanning Electron Microscopy. Degradation study was also performed for the nanofibers. The tensile strength of the aligned nanofibers was measured via Dynamic Mechanical Analysis (DMA). Before functionalization of the aligned nanofibers, Schwann cells were seeded on the constructed scaffolds and the biocompatibility of the aligned nanofibers were investigated as pre-cell culture study. Cell proliferation studies were achieved and alignment of the cells on aligned nanofibers were visualized via Confocal microscopy. Therefore, it was proven by these studies that the directed nanofibers provided the alignment of the Schwann cells on the scaffolds. After the characterization of the scaffold, the peptide functionalization of the scaffold was achieved via EDC/NHS cross-linking. In order to prove the peptide binding, the peptides were labeled via fluorescent tag. The nanofibers were visualized via confocal microscopy and the homogenous distribution of peptides on nanofibers was observed. In order to prove that the constructed scaffold may have the potential to trigger regeneration and axon extension, rat bone marrow mesenchymal stem cells (rMSCs)were seeded on functionalized nanofibers and the effects of the peptide binding to the scaffold were investigated via in vitro studies. MTS cell proliferation assay was used to check the biocompatibility of the scaffold with the stem cells. The cells were also tagged with fluorescent neural markers and confocal microscopy was performed to check the differentiation of rMSCs. The mRNA levels of the different target genes were also investigated to prove differentiation capacity via Real Time PCR application. As a conclusion of the study, it was proven that PLLA/PBSu blend is convenient for scaffold construction both for sponge structures and aligned nanofibers. The neural cells adapted the scaffold successfully. Especially, the aligned nanofibers are promising to be used in neural tissue damages after functionalized by neuroactive peptides and seeded with MSCs as they triggered neural differentiation of MSCs. To sum up, the proposed design can be an alternative scaffold for treatment in neural tissue engineering according to the in vitro investigations.