(267d) Development of Gelatin and Graphene Based Conduits Using 3D Printing Strategies for the Transdifferentiation of Mesenchymal Stem Cells into Schwann Cell-like Phenotypes through Electrical Stimuli

Transdifferentiated mesenchymal stem cells (MSCs) possessing Schwann Cell (SC) like phenotypes have recently been transplanted in different conduits for peripheral nerve regeneration to enable autologous transplants for patients with peripheral nerve damage. However, current difficulties in controlling the final fate of the implanted cell population and providing scalable differentiation protocols along with an ideal 3D scaffold completely mimicking the extracellular matrix limit the clinical use of these transdifferentiated MSCs. In addition, the current knowledge of the effect of mechanical and structural properties of 3D scaffolds on the differentiation behavior of MSCs is limited.¹

In our previous study², we developed gelatin based 3D conduits possessing different mechanical properties and microstructures for the chemical transdifferentiation of MSCs into SC-like phenotypes. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits provided the most favorable environment for MSC transdifferentiation to SC-like phenotypes. Our results indicated that the 3D ladder-like and macroporous structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks. The 3D ladder-like conduit structure with complex modulus of ~0.4x10⁶ Pa and pore size of ~150 nm provided the most favorable microenvironment for MSC differentiation leading to ~ 85% immunolabeling of all SC markers (α-S100, α-S100β and α-p75). The transdifferentiated MSCs in ladder-like 3D conduits also secreted significant amounts of neurotrophic growth factors (NGF: 2.5 pg/mL and GDNF: 0.7 pg/mL per cell). This work demonstrated the importance of controlling the 3D microstructure and mechanical properties to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.²

The procedures regularly applied for the transdifferentiation of MSCs to SC-like phenotypes requires the use of high cost chemicals or growth factors. Our recent work showed that graphene-based materials can serve as an effective alternative to transdifferentiate MSCs in to SC-like phenotypes using electrical stimuli. We observed significant enhancement in MSCs transdifferentiation (~85% of the MSCs differentiated into SCs-like phenotypes) and paracrine activity (differentiated MSCs secreted ~80 ng/mL NGF, 10 ng/mL BDNF and GDNF) via electrical stimuli as compared to the conventional chemical differentiation treatment (~75% of the MSCs differentiated into SCs like phenotypes and ~55 ng/mL of NGF secretion) using graphene based materials.³

Based on this background, in this study, gelatin and graphene based conduits possessing different microstructures and mechanical properties were fabricated through 3D printing. The effect of 3D conduit microstructure and mechanical properties along with the applied electrical stimuli on MSCs behavior and transdifferentiation into SC-like phenotypes were investigated. The results indicated that the application of electrical stimuli through the conductive graphene layers within the gelatin based 3D microstructure provided a profound effect on the MSCs to SC-like phenotype differentiation and paracrine activity. We observed significant SC marker staining and enhanced NGF secretion (5.2 pg/mL per cell) compared to our previous works. These results suggest that the electrical stimuli applied within the 3D gelatin matrix enables enhanced differentiation and paracrine activity compared to transdifferentiation procedures involving electrical stimuli applied in 2D substrates³ and chemical stimuli applied in 3D gelatin scaffolds², leading to promising nerve regeneration strategies.

References:

1. Elizabeth J Sandquist, Metin Uz, Anup D Sharma, Bhavika B Patel, Surya K Mallapragada, Donald S Sakaguchi, Stem Cells, Bioengineering, and 3-D Scaffolds for Nervous System Repair and Regeneration, Neural Engineering 2016, 25-81.

2. Uz, M.; Büyüköz, M.; Sharma, A. D.; Sakaguchi, D. S.; Altinkaya, S. A.; Mallapragada, S. K., Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes. Acta Biomaterialia 2017.

3. Das, S. R.*; Uz, M.*; Ding, S.; Lentner, M. T.; Hondred, J. A.; Cargill, A. A.; Sakaguchi, D. S.; Mallapragada, S.; Claussen, J. C., Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits. Advanced Healthcare Materials 2017, 6 (7). (* indicates equal contribution)