Lokesh Karthik Narayanan

Moisture absorption into hygroscopic/hydrophilic materials used in fused deposition modeling (FDM) can diminish desired mechanical properties. Sensitivity to moisture is dependent on material properties and environmental factors and needs characterization. In this article, moisture sensitivity of four grades of polylactic acid (PLA) filaments and four different ratios of PLA/polybutylene succinate (PBS) blended filaments were characterized through FDM printed American society for testing and materials (ASTM-D638) test samples after conditioning the filaments at different relative humidity levels. The tensile testing and scanning electron microscopy (SEM) of the samples' fracture surfaces revealed that PLA 4043D was the most moisture-sensitive among the chosen grades of PLA filaments. Through filament tension test and melt flow index (MFI) testing it was observed that moisture had a significant detrimental effect (20% reduction in tensile strength and 50% increase in MFI) on PLA 4043D filaments. Samples from moisture-conditioned PLA/PBS 75/25 blended filaments displayed a significant reduction (10%) in tensile strength. Moreover, the MFI of 75/25 filaments was increased with subsequent increases in moisture level. Investigation of tensile properties of ASTM samples made from four grades of PLA filaments exposed to room temperature and humidity conditions for 3 months showed an even more significant decrease in strength (ranging from 24% to 36%).

The use of fused deposition modeling (FDM) in printing polymers for various applications has been ever increasing. However, its utilization in printing polymers for high-strength and superior surface finish applications is still a challenge, primarily due to process intrinsic defects, i.e., voids between the layers and the rough exterior arising from unrestrained deposition of molten polymer. This research hypothesizes that application of ultrasonic vibration (USV) post-fabrication could minimize these shortcomings. For this investigation, ASTM D638 Type IV samples were FDM-printed using poly(lactic) acid (PLA). Through screening experiments, an optimized set of ultrasonic parameters was determined. Then, the effect of both-sided ultrasonic application was characterized. Subsequently, the impact of USV on the samples’ physical, tensile, and morphological properties was examined by varying the layer height, infill patterns, and % infill density. Up to 70% roughness reduction was observed as a result of post-FDM ultrasonic application. Additionally, the tensile strength of the samples increased by up to 15.31%. Moreover, for some lower % infill samples, post-ultrasonic tensile strengths were higher than 100% infill control samples. Analysis of scanning electron microscopy (SEM) and X-ray computed tomography (CT) imagery indicated enhanced layer consolidation and reduced void presence in samples treated with ultrasonic. The combination of ultrasonic-generated heat and downward pressure promoted a synergistic squeeze flow and intermolecular diffusion across consecutive layers of polymers. As a result, increased tensile strength and surface finish were achieved while dimensional change was marginal.

The use of fused deposition modeling (FDM) in printing polymers for various applications has been ever increasing. However, its utilization in printing polymers for high-strength and superior surface finish applications is still a challenge, primarily due to process intrinsic defects i.e., voids between the layers and the rough exterior arising from unrestrained deposition of molten polymer. This research hypothesizes that application of ultrasonic vibration (USV) post-fabrication could minimize these shortcomings. For this investigation, ASTM D638 Type IV samples were FDM printed using Poly(lactic) Acid (PLA). Through screening experiments, an optimized set of ultrasonic parameters was determined. Then, the effect of both-sided ultrasonic application was characterized. Subsequently, the impact of USV on the samples’ physical, tensile, and morphological properties was examined by varying the layer height, infill patterns, and % infill density. Up to 70% roughness reduction was observed as a result of post-FDM ultrasonic application. Additionally, the tensile strength of the samples increased by up to 15.31%. Moreover, for some lower % infill samples, post-ultrasonic tensile strengths were higher than 100% infill control samples. Analysis of scanning electron microscopy (SEM) and X-ray computed tomography (CT) imagery indicated enhanced layer consolidation and reduced void presence in samples treated with ultrasonic. The combination of ultrasonic-generated heat and downward pressure promoted a synergistic squeeze flow and intermolecular diffusion across consecutive layers of polymers. As a result, increased tensile strength and surface finish were achieved while dimensional change was marginal.

In this study we characterized the process–structure interactions in melt extrusion-based 3D bioplotting of polycaprolactone (PCL) and developed predictive models to enable the efficient design and processing of scaffolds for tissue engineering applications. First, the effects of pneumatic extrusion pressure (0.3, 0.4, 0.5, 0.6 N/mm²), nozzle speed (0.1, 0.4, 1.0, 1.4 mm/s), strand lay orientation (0°, 45°, 90°, 135°), and strand length (10, 20, 30 mm) on the strand width were investigated and a regression model was developed to map strand width to the two significant parameters (extrusion pressure and nozzle speed; p < 0.05). Then, proliferation of NIH/3T3 fibroblast cells in scaffolds with two different stand widths fabricated with different combinations of the two significant parameters was assessed over 7 days, which showed that the strand width had a significant effect on proliferation (p < 0.05). The effect of strand lay orientation (0° and 90°) on tensile properties of non-porous PCL specimens was determined and was found to be significantly higher for specimens with 0° lay orientation (p < 0.05). Finally, these data were used to develop and experimentally validate a finite element model for a porous PCL specimen with 1:1 ratio of inter-strand spacing to strand width.

Biofabrication processes can affect biological quality attributes of encapsulated cells within constructs. Currently, assessment of the fabricated constructs is performed offline by subjecting the constructs to destructive assays that require staining and sectioning. This drawback limits the translation of biofabrication processes to industrial practice. In this work, we investigate the dielectric response of viable cells encapsulated in bioprinted 3D hydrogel constructs to an applied alternating electric field as a label-free non-destructive monitoring approach. The relationship between β -dispersion parameters (permittivity change—Δ ε , Cole–Cole slope factor— α , critical polarization frequency— f _c ) over the frequency spectrum and critical cellular quality attributes are investigated. Results show that alginate constructs containing a higher number of viable cells (human adipose derived stem cells—hASC and osteosarcoma cell line—MG63) were characterized by significantly higher Δ ε and α (both p < 0.05). When extended to bioprinting, results showed that changes in hASC proliferation and viability in response to changes in critical bioprinting parameters (extrusion pressure, temperature, processing time) significantly affected ∆ ε , α , and f _c . We also demonstrated monitoring of hASC distribution after bioprinting and changes in proliferation over time across the cross-section of a bioprinted medial knee meniscus construct. The trends in ∆ ε over time were in agreement with the alamarBlue assay results for the whole construct, but this measurement approach provided a localized readout on the status of encapsulated cells. The findings of this study support the use of dielectric impedance spectroscopy as a label-free and non-destructive method to characterize the critical quality attributes of bioprinted constructs.

In any three dimensional (3D) biofabrication process, assessing critical biological quality attributes of 3D constructs such as viable cell number, cell distribution and metabolic activity is critical to determine the suitability and success of the process. One major limitation in current state-of-the-art is the lack of appropriate methods to monitor these quality attributes in situ in a non-destructive, label-free manner. In this study, we investigate the feasibility of using dielectric impedance spectroscopy to address this gap. We first measured the relative permittivity of 3D alginate constructs with four different concentrations of encapsulated MG63 cells (1–6.5 million cells/mL) and found them to be statistically significantly different (p < 0.05). Within the tested range, the relationship between cell concentration and relative permittivity was noted to be linear (R² = 0.986). Furthermore, we characterized the β-dispersion parameters for MG63-encapsulated in alginate (6.5 million cells/mL). These results demonstrate that dielectric impedance spectroscopy can be used to monitor critical quality attributes of cell-encapsulated 3D constructs. Owing to the measurement efficiency and non-destructive mode of testing, this method has tremendous potential as an in-process quality control tool for 3D biofabrication processes and the long-term monitoring of cell-encapsulated 3D constructs.

Bioinks play a central role in 3D-bioprinting by providing the supporting environment within which encapsulated cells can endure the stresses encountered during the digitally-driven fabrication process, and continue to mature, proliferate, and eventually form extracellular matrix (ECM). In order to be most effective, it is important that bioprinted constructs recapitulate the native tissue milieu as closely as possible. As such, musculoskeletal soft tissue constructs can benefit from bioinks that mimic their nanofibrous matrix constitution, which is also critical to their function. This study focuses on the development and proof-of-concept assessment of a fibrous bioink composed of alginate hydrogel, polylactic acid nanofibers and human adipose-derived stem cells (hASC) for bioprinting such tissue constructs. First, hASC proliferation and viability were assessed in 3D-bioplotted strands over 16 days in vitro. Then, a human medial knee meniscus digitally modeled using magnetic resonance images was bioprinted and evaluated over 8 weeks in vitro. Results show that the nanofiber-reinforced bioink allowed higher levels of cell proliferation within bioprinted strands, with a peak at day 7, while still maintaining a vast majority of viable cells at day 16. The cell metabolic activity on day 7 was 28.5% higher in this bioink compared to the bioink without nanofibers. Histology of the bioprinted meniscus at both 4 and 8 weeks showed 54% and 147% higher cell density, respectively, in external versus internal regions of the construct. Presence of collagen and proteoglycans was also noted in areas surrounding the hASC, indicating ECM secretion and chondrogenic differentiation.

Scaffolds play an important role in tissue engineering by providing structural framework and a surface for cells to attach, proliferate, and secrete extracellular matrix (ECM). In order to enable efficient tissue formation, delivering sufficient cells into the scaffold three-dimensional (3D) matrix using traditional static and dynamic seeding methods continues to be a critical challenge. In this study, we investigate a new cell delivery approach utilizing deposition of hydrogel-cell encapsulated microspheroids into polycaprolactone (PCL) scaffolds to improve the seeding efficiency. Three-dimensional-bioplotted PCL constructs (0 deg/90 deg lay down, 284 ± 6 μm strand width, and 555 ± 8 μm strand separation) inoculated with MG-63 model bone cells encapsulated within electrostatically generated calcium-alginate microspheroids (Ø 405 ± 13 μm) were evaluated over seven days in static culture. The microspheroids were observed to be uniformly distributed throughout the PCL scaffold cross section. Encapsulated cells remained viable within the constructs over the test interval with the highest proliferation noted at day 4. This study demonstrates the feasibility of the new approach and highlights the role and critical challenges to be addressed to successfully utilize 3D-bioprinting for microencapsulated cell delivery.