Chapter Three: Materials and Methods Chapter Three Materials and Methods 54 Chapter Three: Materials and Methods 3.1 Polymer Synthesis All the polymers used in this study were synthesized at the Centre de Recherche sur les Biopolymères Artificiels (UMR CNRS 5473, Montpellier, France). ∈-Caprolactone was purchased from Acros Organics. Ethylene glycol, monohydroxyl PEG (mPEG) and dihydroxyl PEG were supplied by Fluka and used as received. DL-lactide (DL-LA) was obtained from Purac (Gorinchem, Holland) and recrystallized from acetone. Zinc powder was received from Merck. PCL homopolymer, PCL-PEG, PCL-PEG-PCL and PEG-PCL-PLA block copolymers were synthesized by ring-opening polymerization (ROP) of ∈-caprolactone using zinc metal as catalyst as reported elsewhere (Li et al, 1998; Huang et al, 2003). 3.1.1 PCL Homopolymer PCL homopolymer was synthesized by bulk ROP of ∈-caprolactone, using ethylene glycol as initiator and zinc powder as catalyst. ∈-Caprolactone and ethylene glycol with a molar ratio of 700/1 and zinc powder (0.05 wt %) were introduced into a round-bottomed flask. After degassing at room temperature, the flask was sealed under vacuum and allowed to rotate at 140°C for 11 days. 3.1.2 PCL/PEG Block Copolymers Predetermined amounts of ∈-caprolactone, mPEG or dihydroxyl PEG and zinc powder were introduced into a round-bottomed flask. The caprolactone/ethylene oxide or [CL] / [EO] molar ratio was 3/1. Degassing 55 Chapter Three: Materials and Methods was performed at 80°C to homogenize the mixture. After cooling, the flask was sealed under vacuum and allowed to rotate at 140°C for days. 3.1.3 PEG/PCL/PLA Block Copolymers The PEG-PCL-P(DL)LA triblock copolymer was synthesized by stepwise ROP of ε-caprolactone and DL-lactide. In the first step, ε-caprolactone (171 g), mPEG5000 (22 g) and zinc powder (0.05 wt-%) were introduced into a roundbottomed flask. Polymerization was carried out under vacuum at 140°C for days. The resulting hydroxyl-bearing PEG-PCL was used as macroinitiator in the second step for the ROP of DL-lactide. DL-lactide (50 g) was allowed to polymerize in the presence of this PEG-PCL (50 g) and zinc powder (0.05 wt%) under vacuum at 140°C for days. 3.1.4 Purification of Polymers All the polymers were recovered by the dissolution/precipitation method with dichloromethane as solvent and ethanol as nonsolvent, followed by filtration and vacuum drying up to constant weight. PEG-PCL-PLA block copolymer was further washed with cold acetone ([...]... principle of the pycnometer is described in Appendix A 3. 5 .3 Micro-CT 3D Imaging The internal 3D architecture and micro-structural formability of as fabricated scaffolds were studied by Micro-CT The micro-CT was set at 19 micron resolution 2D analyses and 3D reconstructions of core regions of the samples were performed which enabled to calculate the porosity and interconnectivity of the scaffolds 3. 5 .3. 1... ILH and ILV loading, one layer out of every 2 layers of filaments was aligned in the direction of loading For 0/60/120° lay-down pattern in ILH loading, one layer out of every 3 layers of filaments was aligned normal to the direction of loading In ILV loading, one layer out of every 3 layers of filaments was aligned in the direction of loading For 0 /30 /60/90/120/150° lay-down pattern in both ILH and. .. morphology and function 87 Chapter Three: Materials and Methods 3. 9 .3. 1 Phase Contrast Light Microscopy Phase-contrast light microscopy (Leica DM IRB, Germany) was used to examine the establishment of the cell phenotype, intercellular connections, and extracellular matrix production on weekly basis for the entire period of 3 weeks 3. 9 .3. 2 Confocal Laser Microscopy (CLM) The distribution and ratio of viable and. .. avoid creation of isolated voids (Gibson and Ashby, 1997) Based on this phenomenon, various scaffolds with different values of FD were built up The scaffolds were fabricated with two polymers (PCL and PCL-PEG) and a series of FDs (1.0, 1.25 & 1.5 mm) applying the liquefier temperature, extrusion pressure and 69 Chapter Three: Materials and Methods deposition speed of 90°C, 4.0 bars and 30 0 mm/min, respectively... filaments were laid as 0° lines, 60° and 120° lines, and in 0 /30 /60/90/120/150° lay-down pattern the filaments were drawn as 0° lines, 30 ° lines, 60° lines, 90° lines, 120° lines and 150° lines Overview of the toolpath of the scaffold design process is presented in Figure 3. 10 in order 70 Chapter Three: Materials and Methods Solid model Solid model Model 0°-line 0°-line 0°-line 30 °-line 90°-line 60°-line 60°-line... speed values of 1.0, 5.0 & 10.0 mm/min (corresponding to strain rates of 0.0 033 , 0.0167 and 0. 033 /s) The modulus of elasticity called compression stiffness, E (MPa) was calculated as the slope of initial linear portion of the stress-strain curve neglecting any toe region formed due to the initial settling of the specimen Compressive strength at yield σy (MPa) was defined as the intersection of the stress–strain... (i.e from OL) of fabrication process at a crosshead speed of 1 mm/min between two steel platens up to a strain level of approximately 60% The stiffness and yield strength were calculated from the stress–strain curve and evaluated as function of degradation time 84 Chapter Three: Materials and Methods 3. 9 Cell Culture Study To investigate the efficacy of the developed scaffolds for tissue engineering. .. media was changed after every two days and the culture was continued for the period of 3 weeks 3. 9 .3 Evaluation of Cell Performance Synthetic and/ or naturally occurring polymers are important elements in new strategies for producing engineered tissue To select appropriate polymeric scaffold for tissue engineering application, it is important to understand the influence of the polymer type as well as scaffold... Corrugated edge before trimming off, (B): Uniform edge after trimming off 72 Chapter Three: Materials and Methods Step 1 Step 2 Step 3 ILH ILV OL Figure 3. 12: Cutting of scaffold blocks for various analyses Three groups of specimens were labeled namely ILH, ILV and OL representing different alignment of the filaments with respect to the longitudinal axis of each final specimen 3. 5 Scaffold Characterization... Figure 3. 10: Overview of scaffold design toolpath; top: 2-angle pattern, middle: 3- angle pattern and bottom: 6-angle pattern 3. 4.4.2 Preparation of Test Specimens The large porous blocks of 50.0 x 50.0 x 5.0 mm were built as initial bulk scaffolds on a flat plastic platform After completion of fabrication the bulk scaffolds were removed from the plastic platform The corrugated edges (Figure 3. 11) of the . the 3D model 61 Chapter Three: Materials and Methods 3. 4 .3 Scaffold Design Various scaffold architectures can be imprinted by applying various lay-down patterns (0/90°, 0/60/120° and 0 /30 /60/90/120/150°). shown in Figure 3. 4 using appropriate positioning of the robotic control system. The lay-down patterns of 0/90°, 0/60/120° and 0 /30 /60/90/120/150° are also called 2-angle, 3- angle and 6-angle. pressure and 69 Chapter Three: Materials and Methods deposition speed of 90°C, 4.0 bars and 30 0 mm/min, respectively. This set of scaffolds was fabricated to investigate the influence of structural