Chapter Five: Conclusions and Recommendations Chapter Five Conclusions and Recommendations 197 Chapter Five: Conclusions and Recommendations 5.1 Conclusions A desktop robot based rapid prototyping (DRBRP) melt extrusion system was designed and developed in house in collaboration with other members in the research team that allowed design and fabrication of 3D scaffolds for tissue engineering applications. This technique directly extruded the material from its granulated or any other form into 3D scaffold in contrast to traditional FDM system that requires prefabricated filament pre-cursor. Most of the established RP systems mainly focus on a single mode of material and/or scaffold processing. The DRBRP was designed to give the user freedom to process a large variety of polymers and/or scaffolds. Virtually, DRBRP system would allow a wide range of thermoplastic material to process into 3D scaffold with any lay-down pattern. A range of synthetic biopolymers namely, PCL and PCL-based copolymers (PCL-PEG, PCL-PEG-PCL and PEG-PCL-PLA) prepared by ring-opening polymerization, were processed into 3D scaffolds with honeycomb-like architecture, and fully interconnected and controllable pore channel. The copolymer had the potential to modulate hydrophilicity and/or degradability and consequently, the biomechanical properties of the matrices by varying the ratio of the polymer components. The scaffold processing demonstrated in one hand, the efficacy of the DRBRP technique to process a wide range of biopolymers and on the other hand, the feasibility of these polymers to be processed into 3D scaffolds. 198 Chapter Five: Conclusions and Recommendations The morphological characterization (pore size, porosity and interconnectivity measurement) by SEM, ultrapycnometer and micro-CT analyses revealed that the lay-down pattern and filament distance were very effective means to control the pore shape, size and porosity. The 0/90 lay-down pattern produced quadrangular pores, 0/60 pattern resulted in triangular pores, whereas 0/30 pattern formed complex polygonal pores. The pore height (in zdirection) and porosity varied from 500 to 2500µm and 66 to 60%, respectively due to the change of pattern from 0/90 to 0/30. Likewise, by changing filament distance from 1.0 to 1.5 mm the pore width (in x- or ydirection) and porosity varied from 500 to 1000µm and 50 to 66%, respectively. Besides, the process parameters (liquefier temperature, extrusion pressure and deposition speed) were found to have good control over the deposited filament diameter and consequently, the pore size and porosity. A range of filament diameter (375 to 623 µm) and porosity (45 to 75%) was obtained by varying the process parameters. In spite of significant change in filament diameter, pore size and porosity, 100% pore interconnectivity was maintained in all the scaffold structures. Mechanical characterization revealed that different mechanical properties could be achieved by utilizing different materials, while the design parameters of the scaffold remained unchanged. The 0/90 lay-down pattern producing 66% porosity resulted in compressive stiffness, 1% offset yield strength and yield strain of 34.87 MPa, 2.69 MPa and 3.5%, respectively for PCL scaffold, and 29.8 MPa, 2.3 MPa and 2.98%, respectively for PCL-PEG scaffold. Similar to porous characteristics, the lay-down pattern, filament distance and 199 Chapter Five: Conclusions and Recommendations process parameters significantly influenced the mechanical properties that were found to be porosity dependant. The loading direction, strain rate and physiological environment also influenced the mechanical properties of the scaffolds. The variation in strain rate revealed the viscoelastic property of the scaffold, while the various loading direction investigated the anisotropy. The compression test in PBS solution at 37˚C resulted in decrease of mechanical properties. It might be due to the effect of external energy input and/or plasticizing effect because of the presence of moisture. However, the decrease was less significant than that was reported by other groups for foam-like scaffolds made of copolymers of PGA/PLA. In vitro degradation study demonstrated that both PCL and PCL-PEG scaffolds realized homogeneous hydrolytic degradation via surface erosion resulting in a consistent and predictable mass and material loss. The linear mass loss caused uniform and linear increase in porosity that accorded with the decrease in mechanical properties. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and degradability of the PCL-PEG copolymer. However, the architectural variation did not influence the degradation kinetics. In reality, accelerated degradation study does not provide conclusive information that would occur in the actual physiological environment rather this procedure enables the comparison of degradability among different scaffolds and materials in a more acceptable time frame. 200 Chapter Five: Conclusions and Recommendations The biocompatibility as well as the ability of the PCL and PCL-PEG scaffolds to favour cell adhesion and function was evaluated successfully using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation and extracellular matrix production on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix. PCL-PEG copolymer scaffolds showed overall better performance in cell culture studies than the PCL homopolymer scaffold that was reflected by the DNA quantification assay. However, the variation in laydown pattern did not significantly influence the cell culture performance. In summary, the results suggest that a scaffold family can be developed with a range of morphological and biomechanical properties by various selections of design and process parameters in combination with different polymers using the in house built DRBRP technique. 201 Chapter Five: Conclusions and Recommendations 5.2 Recommendations This PhD thesis was the part of a research program that aims to establish a scaffold library that would have a database on the physical and biomechanical characteristics of various scaffolds in relation to different materials and designs. This data produced from this thesis would provide the tissue engineer with guide line to develop scaffolds as required for the tissue engineering applications. Accordingly, some of the areas that deserve further research are listed below: Physical and biomechanical characterization of the scaffolds that should be fabricated using various nozzle sizes, mixed architectures in the same structure and laying down multiple layers with same angle orientation. Incorporation of bioactive agents, like hydroxyapatite (HA) or tri-calcium phosphate (TCP) in the scaffold material to produce osteoconductive scaffolds with adequate stiffness and strength for high load-bearing applications. Extensive qualitative and quantitative cell culture studies using human cells. In vivo study that investigates the degradation and resorption kinetics of scaffold-tissue constructs in regard to foreign body reactions using animal models. Some predictive models (e.g. model for scaffold porosity based on extruding materials and process parameters, model for degradation kinetics and/or cell behavior) can be developed and applied to correlate the compressive characteristics with the hatch pattern. 202 . Chapter Five: Conclusions and Recommendations Chapter Five Conclusions and Recommendations 197 Chapter Five: Conclusions and Recommendations 5. 1 Conclusions. namely, PCL and PCL-based copolymers (PCL-PEG, PCL-PEG-PCL and PEG-PCL-PLA) prepared by ring-opening polymerization, were processed into 3D scaffolds with honeycomb-like architecture, and fully. diameter and consequently, the pore size and porosity. A range of filament diameter (3 75 to 623 µm) and porosity ( 45 to 75% ) was obtained by varying the process parameters. In spite of significant