Effects of Shear Stress Gradients on Ewing Sarcoma Cells Using 3D Printed Scaffolds and Flow Perfusion.

In this work, we combined three-dimensional (3D) scaffolds with flow perfusion bioreactors to evaluate the gradient effects of scaffold architecture and mechanical stimulation, respectively, on tumor cell phenotype. As cancer biologists elucidate the relevance of 3D in vitro tumor models within the drug discovery pipeline, it has become more compelling to model the tumor microenvironment and its impact on tumor cells. In particular, permeability gradients within solid tumors are inherently complex and difficult to accurately model in vitro. However, 3D printing can be used to design scaffolds with complex architecture, and flow perfusion can simulate mechanical stimulation within the tumor microenvironment. By modeling these gradients in vitro with 3D printed scaffolds and flow perfusion, we can identify potential diffusional limitations of drug delivery within a tumor. Ewing sarcoma (ES), a pediatric bone tumor, is a suitable candidate to study heterogeneous tumor response due to its demonstrated shear stress-dependent secretion of ligands important for ES tumor progression. We cultured ES cells under flow perfusion conditions on poly(propylene fumarate) scaffolds, which were fabricated with a distinct pore size gradient via extrusion-based 3D printing. Computational fluid modeling confirmed the presence of a shear stress gradient within the scaffolds and estimated the average shear stress that ES cells experience within each layer. Subsequently, we observed enhanced cell proliferation under flow perfusion within layers supporting lower permeability and increased surface area. Additionally, the effects of shear stress gradients on ES cell signaling transduction of the insulin-like growth factor-1 pathway elicited a response dependent upon the scaffold gradient orientation and the presence of flow-derived shear stress. Our results highlight how 3D printed scaffolds, in combination with flow perfusion in vitro, can effectively model aspects of solid tumor heterogeneity for future drug testing and customized patient therapies.

Extrusion-based 3D printing of poly(propylene fumarate) scaffolds with hydroxyapatite gradients.

The primary focus of this work is to present the current challenges of printing scaffolds with concentration gradients of nanoparticles with an aim to improve the processing of these scaffolds. Furthermore, we address how print fidelity is related to material composition and emphasize the importance of considering this relationship when developing complex scaffolds for bone implants. The ability to create complex tissues is becoming increasingly relevant in the tissue engineering community. For bone tissue engineering applications, this work demonstrates the ability to use extrusion-based printing techniques to control the spatial deposition of hydroxyapatite (HA) nanoparticles in a 3D composite scaffold. In doing so, we combined the benefits of synthetic, degradable polymers, such as poly(propylene fumarate) (PPF), with osteoconductive HA nanoparticles that provide robust compressive mechanical properties. Furthermore, the final 3D printed scaffolds consisted of well-defined layers with interconnected pores, two critical features for a successful bone implant. To demonstrate a controlled gradient of HA, thermogravimetric analysis was carried out to quantify HA on a per-layer basis. Moreover, we non-destructively evaluated the tendency of HA particles to aggregate within PPF using micro-computed tomography (µCT). This work provides insight for proper fabrication and characterization of composite scaffolds containing particle gradients and has broad applicability for future efforts in fabricating complex scaffolds for tissue engineering applications.

3D printing PLGA: a quantitative examination of the effects of polymer composition and printing parameters on print resolution.

In the past few decades, 3D printing has played a significant role in fabricating scaffolds with consistent, complex structure that meet patient-specific needs in future clinical applications. Although many studies have contributed to this emerging field of additive manufacturing, which includes material development and computer-aided scaffold design, current quantitative analyses do not correlate material properties, printing parameters, and printing outcomes to a great extent. A model that correlates these properties has tremendous potential to standardize 3D printing for tissue engineering and biomaterial science. In this study, we printed poly(lactic-co-glycolic acid) (PLGA) utilizing a direct melt extrusion technique without additional ingredients. We investigated PLGA with various lactic acid:glycolic acid (LA:GA) molecular weight ratios and end caps to demonstrate the dependence of the extrusion process on the polymer composition. Micro-computed tomography was then used to evaluate printed scaffolds containing different LA:GA ratios, composed of different fiber patterns, and processed under different printing conditions. We built a statistical model to reveal the correlation and predominant factors that determine printing precision. Our model showed a strong linear relationship between the actual and predicted precision under different combinations of printing conditions and material compositions. This quantitative examination establishes a significant foreground to 3D print biomaterials following a systematic fabrication procedure. Additionally, our proposed statistical models can be applied to couple specific biomaterials and 3D printing applications for patient implants with particular requirements.

Extrusion-Based 3D Printing of Poly(propylene fumarate) in a Full-Factorial Design.

3D printing has emerged as an important technique for fabricating tissue engineered scaffolds. However, systematic evaluations of biomaterials for 3D printing have not been widely investigated. We evaluated poly(propylene fumarate) (PPF) as a model material for extrusion-based printing applications. A full-factorial design evaluating the effects of four factors (PPF concentration, printing pressure, printing speed, and programmed fiber spacing) on viscosity, fiber diameter, and pore size was performed layer-by-layer on 3D scaffolds. We developed a linear model of printing solution viscosity, where concentration of PPF had the greatest effect on viscosity, and the polymer exhibited shear thinning behavior. Additionally, linear models of pore size and fiber diameter revealed that fiber spacing and pressure had the greatest effect on pore size and fiber diameter, respectively, but interplay among the factors also influenced scaffold architecture. This study serves as a platform to determine if novel biomaterials are suitable for extrusion-based 3D printing applications.

Pre-clinical characterization of tissue engineering constructs for bone and cartilage regeneration.

Pre-clinical animal models play a crucial role in the translation of biomedical technologies from the bench top to the bedside. However, there is a need for improved techniques to evaluate implanted biomaterials within the host, including consideration of the care and ethics associated with animal studies, as well as the evaluation of host tissue repair in a clinically relevant manner. This review discusses non-invasive, quantitative, and real-time techniques for evaluating host-materials interactions, quality and rate of neotissue formation, and functional outcomes of implanted biomaterials for bone and cartilage tissue engineering. Specifically, a comparison will be presented for pre-clinical animal models, histological scoring systems, and non-invasive imaging modalities. Additionally, novel technologies to track delivered cells and growth factors will be discussed, including methods to directly correlate their release with tissue growth.

Technical Report: Correlation Between the Repair of Cartilage and Subchondral Bone in an Osteochondral Defect Using Bilayered, Biodegradable Hydrogel Composites.

The present work investigated correlations between cartilage and subchondral bone repair, facilitated by a growth factor-delivering scaffold, in a rabbit osteochondral defect model. Histological scoring indices and microcomputed tomography morphological parameters were used to evaluate cartilage and bone repair, respectively, at 6 and 12 weeks. Correlation analysis revealed significant associations between specific cartilage indices and subchondral bone parameters that varied with location in the defect (cortical vs. trabecular region), time point (6 vs. 12 weeks), and experimental group (insulin-like growth factor-1 only, bone morphogenetic protein-2 only, or both growth factors). In particular, significant correlations consistently existed between cartilage surface regularity and bone quantity parameters. Overall, correlation analysis between cartilage and bone repair provided a fuller understanding of osteochondral repair and can help drive informed studies for future osteochondral regeneration strategies.

Open-source three-dimensional printing of biodegradable polymer scaffolds for tissue engineering.

The fabrication of scaffolds for tissue engineering requires elements of customization depending on the application and is often limited due to the flexibility of the processing technique. This investigation seeks to address this obstacle by utilizing an open-source three-dimensional printing (3DP) system that allows vast customizability and facilitates reproduction of experiments. The effects of processing parameters on printed poly(ε-caprolactone) scaffolds with uniform and gradient pore architectures have been characterized with respect to fiber and pore morphology and mechanical properties. The results demonstrate the ability to tailor the fiber diameter, pore size, and porosity through modification of pressure, printing speed, and programmed fiber spacing. A model was also used to predict the compressive mechanical properties of uniform and gradient scaffolds, and it was found that modulus and yield strength declined with increasing porosity. The use of open-source 3DP technologies for printing tissue-engineering scaffolds provides a flexible system that can be readily modified at a low cost and is supported by community documentation. In this manner, the 3DP system is more accessible to the scientific community, which further facilitates the translation of these technologies toward successful tissue-engineering strategies.

Dual growth factor delivery from bilayered, biodegradable hydrogel composites for spatially-guided osteochondral tissue repair.

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair.