The Development of Three-dimensional Polymeric Assembly for Neural Tissue Engineering Applications

三維聚合物組裝材料在神經組織工程中的應用

Student thesis: Doctoral Thesis

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Award date19 Aug 2019

Abstract

A great challenge in tissue engineering is to generate tissue constructs with high biological activity, high structural stability and physiological relevance, to recapitulate the characteristics of native tissues. Although efforts have been made to fabricate engineered tissues by depositing cell-laden extracellular matrix (ECM) within the synthetic tubal networks, it is still challenging to develop a thick vascularized in vitro tissue model. Here, a novel freeze-coating approach is developed to fabricate 3D microfluidic tubal networks via a sacrificial 3D printing process. For enabling nutrient delivery into the core of the tissue model, the wall of perfusable channels within the microfluidic tubal networks can be adjusted into a microporous structure with the addition of DMSO into the coating solution. Besides, the sacrificial hydrogels used in this thesis could be removed as required, and the tissue model has high structural integrity due to the high mechanical stability of the microfluidic tubal network. The 3D tubal network is able to be functionalized with different chemicals and proteins to improve its biocompatibility and cell affinity.

To develop 3D thick vascularized neural constructs with blood-brain-barrier (BBB) for shuttle peptide and drug evaluation, the 3D microfluidic tubal network is endothelialized and replete with cell-laden hydrogels. The microfluidic tubal network is mechanically stable to maintain the integrity of the BBB model. The engineered neural construct consists of neural stem cell-laden collagen, and PCL/PLGA microfluidic tubal networks, on which endothelial cells, astrocytes and pericytes are located spatially around the luminal and abluminal sides of perfusable channels, forming a brain miniature with BBB-like barrier. Notably, the perfusion of the medium in the endothelialized microfluidic tubal network with BBB function is able to support neuronal differentiation. Moreover, the BBB-like barrier exhibits a high expression level of tight junction proteins and selective permeability for shuttle peptides and drugs. The effect of a BBB-penetrating drug on neurite outgrowth is also studied in the model.

For practical applications of neural tissue engineering in vivo, we develop an injectable cell delivery-system based on a modified gelatin matrix integrated with shape-memory polymer fibers which were used to load embryonic stem cell (ESC) derived motor neurons for minimally invasive treatment of the spinal cord injury. The composite-hydrogel is based on a modified gelatin matrix integrated with shape-memory polymer fibers. The gelatin-matrix creates a local microenvironment for cell assembly and also acts as a lubricant during injection through a fine catheter. Notably, shape-memory nanomesh is able to recover to maintain the microstructures even after dramatic deformation from injection operation, providing the necessary support and guidance for motor neuron differentiation. We find that the composite-hydrogel with an aligned fiber scaffold greatly improves the viability of ESCs and their differentiation towards motor neurons in a three-dimensional way in vivo. When transplanted to SCI (spinal cord injury) animals by injection, the mESC loaded composite hydrogels are identified to enhance tissue regeneration and motor function recovery in mice significantly. With this proof-of-concept study, we believe that the injectable composite-hydrogel system provides a promising solution for in vivo cell delivery with minimum invasiveness and can be readily extended to other stem-cell-based regenerative treatments.

In summary, the thesis integrated biocompatible synthetic polymer and cellular matrix to fabricate a 3D microfluidic tubal network for the construction of vascularized tissue model, and demonstrated the successful application of a composite hydrogel matrix with shape memory electrospun fibers as an excellent cell delivery system to treat spinal cord injury. This thesis provides novel insight about the role of vascularized brain model in drug evaluation and the injectable composite hydrogel system in the stem cell-based treatment of spinal cord injury.