Development and in vitro characterization of three dimensional biodegradable scaffolds for peripheral nerve tissue engineering

Abstract

Tissue engineering emerges nowadays to seek new solutions to damaged tissues and/or organs by replacing or repairing them with engineered constructs or scaffolds. In nerve tissue engineering, scaffolds for the repair of peripheral nerve injuries should act to support and promote axon growth following implantation. It is believed that substantial progress can be made by creating scaffolds from biomaterials, with growth-promoting molecules and spatially-controlled microstructure. To this end, this research aims to develop three dimensional (3D) scaffolds for peripheral nerve tissue regeneration by focusing on studies on the axon guidance, development and characterization of a novel 3D scaffold, and visualization of scaffolds by means of synchrotron-based diffraction enhanced imaging (DEI). Axon guidance is one of crucial considerations in developing of nerve scaffolds for nerve regeneration. In order to study the axon guidance mechanism, a two dimensional (2D) grid micropatterns were created by dispensing chitosan or laminin-blended chitosan substrate strands oriented in orthogonal directions; and then used in the in vitro dorsal root ganglion (DRG) neuron culture experiments. The results show the effect of the micropatterns on neurite directional growth can preferentially grow upon and follow the laminin-blended chitosan pathways. A novel 3D scaffold was developed for potential applications to peripheral nerve tissue engineering applications. The scaffolds were fabricated from poly L-lactide (PLLA) mixed with chitosan microspheres (CMs) by using a rapid freeze prototyping (RFP) technique, allowing for controllable scaffold microstructure and bioactivities protein release. The scaffold characterization shows that (1) the mechanical properties of the scaffolds depend on the ratio of CMs to PLLA as well as the cryogenic temperature and (2) the protein release can be controlled by adjusting the crosslink degree of the CMs and prolonged after the CMs were embedded into the PLLA scaffolds. Also, the degradation properties of the scaffolds were investigated with the results showing that the addition of CMs to PLLA can decrease the degradation rate as compared to pure PLLA scaffolds. This allows for another means to control the degradation rate. Visualization of polymer scaffolds in soft tissues is challenging, yet essential, to the success of tissue engineering applications. The x-ray diffraction enhanced imaging (DEI) method was explored for the visualization of the PLLA/CMs scaffolds embedded in soft tissues. Among various methods examined, including conventional radiography and in-line phase contrast imaging techniques, the DEI was the only technique able to visualize the scaffolds embedded in unstained muscle tissue as well as the microstructure of muscle tissue. Also, it has been shown that the DEI has the capacity to image the scaffolds in thicker tissue, and reduce the radiation doses to tissues as compared to conventional radiography. The methods and results developed/obtained in this study represent a substantial progress in the development and characterization of 3D scaffolds. This progress forms a basis for the future tests on the scaffolds as applied for peripheral nerve injuries.