Bioprinting and characterization of medium viscosity alginate scaffold for nerve tissue regeneration
Sarker, MD Ariful Islam
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Injured peripheral nerves with a gap >2mm often demonstrate poor regeneration ability, where scaffolds made from biomaterials are possibly used to bridge the gap for functional recovery. To fabricate such scaffolds, extrusion-based three dimensional (3D) printing technique shows promising due to its ability to precisely extrude biomaterial solution and build 3D structures by a layer-by-layer fashion. However, the 3D printing technique faces several challenges in fabricating scaffolds for nerve tissue regeneration. Among them, the printability, structural integrity, and biological performance of scaffolds printed from sodium alginate (SA) (a biomaterial widely used in nerve tissue regeneration) are the key issues. To address the issues, this thesis aims to develop SA scaffolds for potential application in peripheral nerve regeneration. Three specific objectives are set so as to investigate (1) the effect of fluid (i.e., SA solution) flow behavior, printing parameters, and concentration of ionic crosslinkers on the printability of SA scaffolds, (2) the influence of SA precursor and ionic crosslinker concentrations on the mechanical and biological properties of scaffolds, and (3) the influence of peptide conjugation with SA molecules on the biological performance of the scaffolds for nerve tissue regeneration. The flow rate in the printing process is critical to the scaffold structure and printability. The first part of dissertation is to examine the flow rate of SA solution or precursor extruded through a tapered needle in the scaffold fabrication process by developing a novel model for its representation. Specifically, the flow rate of the medium viscosity SA precursor was modeled by taking into account of both slip and shear flow from a tapered needle. Since the flow rate of SA precursor depends on its flow behavior, model predicting the flow behavior of the hydrogel precursor was also developed from regression of experimental data. For different extrusion pressures (e.g. 20, 25, 30, and 40 kPa) and concentrations (e.g. 2, 3, and 4%) of SA precursor, the flow rate model predicted with reasonable accuracy (coefficient of determination, R2 = 0.98). Further, at various needle diameters (0.2, 0.25, 0.41, and 0.61 mm) and temperatures (25, 35, 45, and 55°C) the flow rate model predicted more accurately for low dispensing pressure (20 kPa, R2=0.99) compared to high pressure (30 kPa, R2=0.98). The mechanical and biological properties of SA scaffold largely depend on the ionic crosslinker used in bioprinting of scaffolds from SA. The second part of dissertation is to conduct a comparative study of three ionic crosslinkers including calcium chloride (CaCl2), barium chloride (BaCl2), and zinc chloride (ZnCl2) on the mechanical and biological properties of 3D printed SA scaffolds. Multiple regression equations were developed to predict the mechanical properties of SA scaffolds; and the printability of SA precursor was evaluated at varying concentrations of both ionic crosslinkers and SA precursor. Experimental results revealed that the elastic modulus of the hydrogels decreasing in the order BaCl2>CaCl2>ZnCl2 over 42 days while Schwann cell (SC) viability decreased in the order CaCl2>BaCl2>ZnCl2 over 7 days. The predictions of multiple regression equations show reasonable agreement with experimental data, while the 3% (w/v) SA precursor demonstrates acceptable printability in CaCl2 and BaCl2 solution. The experimental and predicted results obtained in this part of work would be useful in selecting the appropriate ionic crosslinkers and concentration of SA precursor for bioprinting of tissue scaffolds. Notably, SA precursor lacks of cell binding motifs in the molecular structure, which significantly limits its applications in nerve tissue regeneration. For improvement, the third part of dissertation is to conjugate peptide molecules into SA, resulting in peptide conjugated SA (PCSA) and to further examine the effect of single and composite PCSA scaffolds on axon regeneration in vitro. In particular, a 2% (w/v) SA precursor was conjugated with either arginine-glycine-aspartate (RGD) or tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptides, or mixture of RGD and YIGSR (1:2), and was bioprinted into cuboid structures. The printability of the composite PCSA precursor was evaluated in terms of the strand width, pore geometry, and angle-formation accuracy at varying concentration of CaCl2 (i.e. 50 - 150 mM); and the mechanical stability of scaffolds was examined over 3 weeks in terms of swelling, degradation, and compression modulus; and surface morphology of the degraded scaffolds. Axon regeneration ability of PCSA scaffolds were assessed by quantifying the viability, morphology, amount of secreted brain derived neurotrophic factor (BDNF) of incorporated SCs, and directional neurite outgrowth in a 2D culture. Experimental results reveal that composite PCSA precursor extruded in 50 mM CaCl2 has good printability and that PCSA scaffolds remains porous over 3 weeks with the elastic modulus decreased by ~70%. Also, the results illustrates that composite PCSA scaffolds facilitate better the viability and morphology of SCs, as well as support greater directional neurite outgrowth as compared to those of single PCSA scaffolds. Taken together, the thesis develops methods to fabricate SA and PCSA scaffolds with results illustrating their potential applications in the regeneration of damaged peripheral nerves.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorChen, Daniel; Schreyer , David
CommitteeEames, Brian; Hedayat, Assem; Zhang, Lifeng
Copyright DateAugust 2019