Achieving molecular orientation in thermally extruded 3D printed objects

Poly(DTD DD) (M_w = 108 kDa) was synthesized using previously described methods (Brocchini et al 1997, 1998). Medical grade PLLA, Purasorb PL 18 (Corbion Biomedical, Lenexa, KS), 1-ethyl-3-(3-dimethaylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Sigma-Aldrich), and lyophilized bovine achilles tendon collagen (Worthington Biochemical Corporation, Lakewood, NJ) were used as received.

DSC data were obtained using a Mettler 823e DSC equipment (Mettler Toledo, Columbus, OH). The samples were sealed in an aluminum pan, and the scans were obtained during heat-cool-reheat cycles at 10 °C min^–1.

Poly(DTD DD) was held at 140, 150, and 160 °C in a Melt Indexer (Extrusion Plastomer Model MP200, Tinius Olsen, Horsham, PA). At 0, 1, 2, 4, and 6 h, at least 100 mg poly(DTD DD) was extruded and dissolved in tetrahydrofuran (THF). The samples were analyzed using gel permeation chromatography (Waters Corporation, Milford, MA; 515 HPLC pump, 717plus autosampler and a 410-differential refractometer).

Rheology measurements were performed in a capillary rheometer (Malvern RH 2000) with a 0.5 mm diameter die. The polymers were dried overnight at 45 °C in a vacuum oven and kept desiccated until use. Approximately 15 g of the polymer was packed into the barrel and allowed to reach the test temperature. The polymers were tested in duplicate at shear rates of 10/s to 5000/s in five logarithmic steps. Poly(DTD DD) was tested at 140, 150, and 160 °C. PLLA was tested at 160, 170, 180, 190, and 200 °C.

Fibers produced by conventional melt spinning and drawing were used for comparison. The fibers were extruded using a ¾ inch single screw extruder (Alex James & Associates, Inc., Greenville, SC) through a 1 mm diameter die at 140 °C, and picked up a roller whose speed was adjusted so that the spooled fiber has a diameter of 170 μm. The fibers were then drawn 2.5 X at 55 °C to a final diameter of 100 μm.

3D printing was done on a pneumatic extrusion-based 3D Bioplotter (EnvisionTEC, Dearborn, MI). Unless otherwise specified, strips of polymer were directly printed on a Kapton tape with the platform at room temperature. The printing parameters, extrusion pressure, polymer temperature, nozzle inner diameter, and printing speed were varied to achieve the best possible orientation (figure 1). Optimum orientation was obtained at 9 bar (max. possible) pressure, at 160 °C, using a 400 μm nozzle, and print speeds of 12 mm s⁻¹. Some of this work was performed using 'mid-air printing' in which the printhead was positioned at a relatively large vertical distance (600 μm) from the printing platform to allow for much higher print speeds.

Zoom In Zoom Out Reset image size

The degree of polymer chain orientation (molecular orientation) of the fibers and 3D printed strips was measured using x-ray diffraction (XRD). In this method, the sample was placed in the path of an x-ray beam (figure 2(A)). Coherently scattered x-rays from the polymer chains interfere and give an enhanced intensity at an angle 2θ determined by spacing between the polymer chains, the d-spacing (d = λ/(2 sin θ), where λ is the wavelength of the x-rays). If the chains are oriented, the scattering is enhanced perpendicular to the direction of orientation, referred to as the equator (figure 2(B)). The azimuthal spread of this reflection can be quantified by the full-width at half-maximum (FWHM, figure 2(C)). FWHM is a measure of the degree of orientation; with a higher orientation corresponding to a smaller azimuthal spread.

Zoom In Zoom Out Reset image size

Two-dimensional (2D) XRD patterns were recorded with Mo Kα radiation (λ = 0.709 Å) using a Smart APEX detector mounted on a Bruker AXS diffractometer. The 2D patterns were used to obtain the azimuthal intensity distribution of the main equatorial reflection at a scattering angle (2θ) ∼ 9.3°, corresponding to ∼4.4 Å interchain distance. The scan was fit to a Gaussian model in MATLAB (Mathworks) to obtain FWHM (Δϕ) calculated using equation (1) (Murthy 2016):

$$\begin{eqnarray}&&I(\phi )={I}_{b}+{e}^{-\mathrm{ln}\left(2\right)* \tfrac{\phi -{\phi }_{0}}{{\left(\tfrac{{\rm{\Delta }}\phi }{2}\right)}^{2}}}\end{eqnarray} \tag{ 1 }$$

where I(ϕ) is the observed intensity, ϕ is the azimuthal angle, and I_b is the background.

The degree of orientation was also assessed by measuring the thermal shrinkage, which was calculated as the ratio of the length of the fiber before and after immersion in water at 65 °C water for 0.5 to 1 min By heating a polymer above its T_g, the deformation stresses applied during molecular alignment are allowed to relax and the molecules are allowed to return to a random orientation. This causes the sample to shrink in the direction of orientation. This thermal shrinkage provides a direct physical evidence of the orientation. The temperature 65 °C was chosen so that the polymer was exposed to temperatures above the first thermal transition event of 55 °C, but below its final melting point of 100 °C (Bedoui et al 2017).

XRD provides the orientation at the molecular level across small areas. Thermal shrinkage provides an average measure of orientation across a larger sample size (50 mm) than x-rays (1 mm), and reflects the inhomogeneities, local stresses as well molecular alignment.

Poly(DTD DD) meniscal scaffolds were printed at 160 °C and 9 bar with a 500 μm inner diameter nozzle, at printing speeds of 2 and 4.5 mm s⁻¹. 20 g/l lyophilized bovine Achilles tendon collagen was ground and swollen in dilute hydrochloric acid (pH = 2.35). The scaffolds were infused with collagen, frozen, and lyophilized. Scaffolds were cross-linked with 10 mM EDC and 5 mM NHS for 6 h, rinsed three times for 10 min in DI water, one time for 3 h in 100 mM sodium phosphate, and again rinsed for 24 h in DI water.

Scaffolds (n = 4 per group) were hydrated in PBS at room temperature for at least one hour. Each scaffold was loaded into cryogenic freeze clamps (TA Instruments, New Castle, DE) with an 8 mm gage length encompassing the central region. The samples were loaded in tension at a rate of 10 mm min⁻¹ to failure (model 5592; Instron, Canton, MA). Circumferential tensile stiffness and ultimate tensile load were calculated for each sample.

DSC scans of poly(DTD DD) are shown in figure 3. The first heating cycle shows an endothermic transition beginning at 40 °C and ending at 110 °C. The cooling cycle shows a crystallization peak at 42 °C. The reheat cycle shows that the main melting point of the polymer to be 58 °C. These values are typical for this polymer (Bedoui et al 2017).

Zoom In Zoom Out Reset image size

Thermal stability of the polymer was assessed by monitoring the degradation of the polymer over a large temperature range. At 150 and 160 °C, the molecular weight decreased by 35% and 33% at 4 h, and by 52% and 57% at 6 h, respectively (figure 4). The data show that an object can be printed for as long as 2 h with poly(DTD DD) at a temperature as high as 160 °C without significant thermal degradation.

Zoom In Zoom Out Reset image size

To assess the low viscosity regime for high print speeds, rheological properties of the polymer were determined at temperatures above those typically required for fiber drawing. Of particular interest is the viscosity of the polymer at low shear rates for this type of 3D printing. The viscosity of poly(DTD DD) decreased markedly as the temperature was increased from 140 °C to 160 °C (figure 5). At low shear rates, the viscosity decreased by 30%–40% as the temperature was raised from 140 °C to 150 °C, and 70%–80% from 150 °C to 160 °C. Thus, relatively small changes in temperature will result in large changes in maximum extrusion rate. Since the objects need to be printed at high nozzle speeds to achieve significant polymer orientation, these results suggest the nozzle should be operated at the highest possible temperatures to lower the melt viscosity.

Zoom In Zoom Out Reset image size

Rheological characteristics of poly(DTD DD) were compared with those of PLLA. PLLA could not be tested below 180 °C because of the high pressures required to extrude the polymer at these temperatures. The shear viscosity decreased by about a third when the temperature of PLLA was increased from 190 °C to 200 °C (figure 5). The shear viscosity of PLLA at 200 °C matched most closely that of poly(DTD DD) at low shear rates. Therefore, PLLA was printed at 200 °C to emulate 3D printing of poly(DTD DD) (see the discussion).

Thermal analysis data, especially capillary rheometer data, were useful in determining the effect of nozzle size on flowrate and the pressures, for selecting the molecular weight of the polymer, and in choosing temperature limits based on degradability. The extrusion parameters that were first chosen based on the rheometer data were optimized iteratively during 3D printing. 3D printing trials were performed at 9 bar, the maximum rated pressure of the 3D printer. The polymer temperature and nozzle diameter had strong effects on the printing speed needed to orient the polymer chains. Higher temperatures permitted higher printing speeds and increased the adhesion of the polymer to the platform and underlying printed polymer layers. The printing speed is the most significant factor in achieving molecular orientation. Platform temperature had only a small effect on orientation.

Effects of polymer temperature and printing speed were examined by printing test strips with a 400 μm inner diameter nozzle in midair. Lower printing temperatures are expected to produce higher orientation similar to that in fiber drawing. The degree of orientation increased significantly with speed at 140 and 160 °C and was greater than that routinely achieved by fiber drawing (figure 6). Higher speeds were required at higher temperature to achieve the same orientation as at lower temperatures. Thermal shrinkage results confirmed the high degree of orientation of the strips; however, the shrinkage values were 7% and 4% less than those measured in drawn fibers for 140 and 160 °C, respectively. The shrinkage indicates a significant increase in orientation with increasing printing speeds; the trend was linear at both 140 and 160 °C (regression analysis, p < 0.001).

Zoom In Zoom Out Reset image size

The orientated samples used in figure 6 were obtained by printing in midair. Although printing at these heights and speeds is not practical with our equipment, this showed that a continuous stream of polymer can be printed at very fast printing speeds to obtain orientations similar to those of conventionally drawn fibers. 3D printing directly onto the platform at similar speeds resulted in fiber breakages. For practical printing directly onto the platform, the maximum speeds at 140 and 160 °C were found to be 0.5 and 1.7 mm s⁻¹, respectively. To further understand the influence of temperature and printing speed on orientation while printing directly onto the platform at practical printing speeds, poly(DTD DD) was 3D printed at 150 °C and 9 bar with a 400 μm inner diameter nozzle at speeds ranging from 1 to 2.5 mm s⁻¹. There was no significant orientation in XRD patterns (FWHM > 75°) (figure 7). Thermal shrinkage results showed moderate polymer orientation that increased linearly with printing speed (regression analysis, p < 0.001).

Zoom In Zoom Out Reset image size

In order to understand the effect of nozzle diameter on orientation, poly(DTD DD) was printed at 160 °C and 9 bar with a larger 600 μm inner diameter nozzle directly on the printing platform, with speeds of 2 to 10 mm s⁻¹. The increase in orientation was moderate from 2 to 4 mm s⁻¹, with no significant increase as printing speed was increased further to 10 mm s⁻¹ (figures 8(A), (B)). In a follow-up study at higher printing speeds of up to 50 mm s⁻¹, a linear correlation was observed between FWHM and printing speeds of 10 to 50 mm s⁻¹, (figures 8(C), (D)). Thermal shrinkage measurements were carried out to confirm this XRD measurement (figure 8(E)). It should be noted that the speeds with significant orientation could be only used to print rectilinear geometries. Curvilinear geometries required that the printing be done at speeds 10 to 20 mm s⁻¹ to avoid problems such as inability to trace the curve or absence of adhesion to a previously deposited layer.

Zoom In Zoom Out Reset image size

With a 600 μm diameter nozzle, orientations similar to with a 400 μm diameter nozzle could be achieved at ∼7-fold greater printing speeds, i.e., by increasing the speed from 1.7 mm s⁻¹ to 10 to 20 mm s⁻¹. An intermediate nozzle (500 μm inner diameter) was tested to determine if significant orientation could be achieved at practical printing speeds. Poly(DTD DD) strips were printed at 160 °C and 9 bar directly on the printing platform from 2 to 12 mm s⁻¹. There was a linear relationship between orientation and printing speed, as measured by XRD and thermal shrinkage (figure 9). Although the orientation was high (FWHM 39° and thermal shrinkage 78%), it did not match those of drawn fibers. These printing speeds represent practical speeds for rectilinear geometries. Printing speeds need to be reduced to 4–8 mm s⁻¹ for printing object with complex curvilinear geometries, such as a meniscal scaffold, with similar resolutions.

Zoom In Zoom Out Reset image size

The results from small, intermediate, and large nozzles at 160 °C show that printing speed needs to be changed to achieve the same degree of polymer orientation with different nozzle diameter. Orientations with a 500 μm nozzle at 2 to 10 mm s⁻¹ were similar to those from a 600 μm nozzle at 10 to 50 mm s⁻¹. The orientation from 400 μm nozzle was much higher, and in fact higher than that of traditionally drawn fibers. As the nozzle diameter was increased from 400 to 500 to 600 μm, the maximum practical printing speed could also increased from 1.7 to 4–8 to10–20 mm s⁻¹, respectively. Interestingly, the 500 μm nozzle yielded significant orientation at practical printing speeds.

The printing platform temperature had the least effect on orientation. Poly(DTD DD) was 3D printed at 0.5 and 1 mm s⁻¹ and the platform temperature was maintained at room temperature and 8 °C (polymer at 150 °C; extrusion pressure: 6.5 bar; and nozzle inner diameter: 400 μm). Although negligible orientation (>94°) was achieved with the platform at 8 °C at all speeds tested, the orientation increases from 114° to 69° with increasing printing speed at 25 °C. These results were confirmed by the low values for thermal shrinkage in all conditions (figure 10). There were no significant differences among any of the conditions tested (one-way ANOVA, p = 0.18). These results suggest that greater orientation can be achieved with the platform at room temperature, most likely due to a slower cooling rate.

Zoom In Zoom Out Reset image size

Meniscus scaffolds were printed using the previously published designs (Ghodbane et al 2018, Ghodbane et al 2019) except for a minor change so that the radial filaments could be extruded continuously instead of in discrete segments. This way, scaffolds could be reproducibly printed at higher speeds with fewer defects (figure 11). The FWHM was reduced by 52% from 72° to 34° by increasing the speed from 2 to 4.5 mm s⁻¹.

Zoom In Zoom Out Reset image size

The tensile modulus of 4.5 mm s⁻¹ meniscus scaffolds was 2.2 times that of the scaffold printed at 2 mm s⁻¹ (142 ± 49 versus 64 ± 23N mm⁻¹) (p = 0.03). There was a marginal (27%) increase in the ultimate load (105 versus 82 N) (p = 0.14) due to printing speed. The structural integrity of the meniscus deteriorated at higher printing speeds as observed with a meniscus fabricated at 6 mm s⁻¹.