Songwan Jin

Evaluation of the effective diffusivity of a freeform fabricated scaffold using computational simulation.

In scaffold-based tissue engineering, sufficient oxygen and nutrient supply into cells within a scaffold is essential to increase cell viability and the proliferation rate. Generally, oxygen and nutrients reach the cells through the media by diffusion in vitro or in vivo, assuming there is no convection flow through a scaffold with small-sized pores. The scaffold diffusion rate depends mainly on the scaffold pore architecture. Thus, understanding the effect of scaffold pore architecture on the diffusion mechanism is necessary to design an efficient scaffold model. This study proposes a computational method to estimate diffusivity using the finite element analysis (FEA). This method can be applied to evaluate and analyze the effective diffusivity of a freeform fabricated 3D scaffold. The diffusion application module of commercial FEA software was used to calculate the spatial oxygen concentration gradient in a scaffold model medium. The effective diffusivities of each scaffold could be calculated from the oxygen concentration data, which revealed that the scaffold pore architecture influences its effective diffusivity. The proposed method has been verified experimentally and can be applied to design pore architectures with efficient diffusion by increasing our understanding of how the diffusion rate within a scaffold is affected by its pore architecture.

Precise stacking of decellularized extracellular matrix based 3D cell-laden constructs by a 3D cell printing system equipped with heating modules

Three-dimensional (3D) cell printing systems allow the controlled and precise deposition of multiple cells in 3D constructs. Hydrogel materials have been used extensively as printable bioinks owing to their ability to safely encapsulate living cells. However, hydrogel-based bioinks have drawbacks for cell printing, e.g. inappropriate crosslinking and liquid-like rheological properties, which hinder precise 3D shaping. Therefore, in this study, we investigated the influence of various factors (e.g. bioink concentration, viscosity, and extent of crosslinking) on cell printing and established a new 3D cell printing system equipped with heating modules for the precise stacking of decellularized extracellular matrix (dECM)-based 3D cell-laden constructs. Because the pH-adjusted bioink isolated from native tissue is safely gelled at 37 °C, our heating system facilitated the precise stacking of dECM bioinks by enabling simultaneous gelation during printing. We observed greater printability compared with that of a non-heating system. These results were confirmed by mechanical testing and 3D construct stacking analyses. We also confirmed that our heating system did not elicit negative effects, such as cell death, in the printed cells. Conclusively, these results hold promise for the application of 3D bioprinting to tissue engineering and drug development.

Three-dimensional culture and interaction of cancer cells and dendritic cells in an electrospun nano-submicron hybrid fibrous scaffold

An artificial three-dimensional (3D) culture system that mimics the tumor microenvironment in vitro is an essential tool for investigating the cross-talk between immune and cancer cells in tumors. In this study, we developed a 3D culture system using an electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold (NFS). A hybrid NFS containing an uninterrupted network of nano- and submicron-scale fibers (400 nm to 2 µm) was generated by deposition onto a stainless steel mesh instead of an aluminum plate. The hybrid NFS contained multiplanar pores in a 3D structure. Surface-seeded mouse CT26 colon cancer cells and bone marrow-derived dendritic cells (BM-DCs) were able to infiltrate the hybrid NFS within several hours. BM-DCs cultured on PCL nanofibers showed a baseline inactive form, and lipopolysaccharide (LPS)-activated BM-DCs showed increased expression of CD86 and major histocompatibility complex Class II. Actin and phosphorylated FAK were enriched where unstimulated and LPS-stimulated BM-DCs contacted the fibers in the 3D hybrid NFS. When BM-DCs were cocultured with mitoxantrone-treated CT26 cells in a 3D hybrid NFS, BM-DCs sprouted cytoplasm to, migrated to, synapsed with, and engulfed mitoxantrone-treated CT26 cancer cells, which were similar to the naturally occurring cross-talk between these two types of cells. The 3D hybrid NFS developed here provides a 3D structure for coculture of cancer and immune cells.

Endothelial monolayers on collagen-coated nanofibrous membranes: cell-cell and cell-ECM interactions.

Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems.

Comparative Efficacies of Collagen-Based 3D Printed PCL/PLGA/β-TCP Composite Block Bone Grafts and Biphasic Calcium Phosphate Bone Substitute for Bone Regeneration

The purpose of this study was to compare bone regeneration and space maintaining ability of three-dimensional (3D) printed bone grafts with conventional biphasic calcium phosphate (BCP). After mixing polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) in a 4:4:2 ratio, PCL/PLGA/β-TCP particulate bone grafts were fabricated using 3D printing technology. Fabricated particulate bone grafts were mixed with atelocollagen to produce collagen-based PCL/PLGA/β-TCP composite block bone grafts. After formation of calvarial defects 8 mm in diameter, PCL/PLGA/β-TCP composite block bone grafts and BCP were implanted into bone defects of 32 rats. Although PCL/PLGA/β-TCP composite block bone grafts were not superior in bone regeneration ability compared to BCP, the results showed relatively similar performance. Furthermore, PCL/PLGA/β-TCP composite block bone grafts showed better ability to maintain bone defects and to support barrier membranes than BCP. Therefore, within the limitations of this study, PCL/PLGA/β-TCP composite block bone grafts could be considered as an alternative to synthetic bone grafts available for clinical use.

Alginate–marine collagen–agarose composite hydrogels as matrices for biomimetic 3D cell spheroid formation

Hydrogels are prototypical matrices for 3D cell culture, of which alginate hydrogels are extensively used. However, ionic crosslinking agents, such as Ca2+, are required to form alginate hydrogels, but introduce Ca2+-associated cytotoxicity and long-term stability issues. Collagen is a promising biomaterial for 3D cell culture scaffolds primarily due to its biocompatibility. In the present study, the authors designed and fabricated a calcium-free, physically crosslinked, efficient, and bioactive hydrogel composed of alginate, marine collagen, and agarose (AmCA) for use in 3D cell cultures. This AmCA hydrogel was assessed by FTIR, swelling property, scanning electron microscopy, phase contrast microscopy, cell proliferation, cell viability, confocal microscopy, transparency and RT-PCR analyses. The gel was found to exhibit excellent cytocompatibility with various tumor and non-tumor cells, to generate high yields of multicellular spheroids, and to promote cellular activity. Furthermore, the transparency of the AmCA hydrogel suggests it can be used without cell-tracking chemicals in morphological studies of cell cultures. Taken together, it would appear that the described physically crosslinked AmCA hydrogel could provide a novel platform for the development of customizable, transparent, biocompatible, functional, easy-to-produce, and cost-effective scaffolds for use in 3D cultures of various cell types.

Fish Scale Collagen Peptides Protect against CoCl2/TNF-α-Induced Cytotoxicity and Inflammation via Inhibition of ROS, MAPK, and NF-κB Pathways in HaCaT Cells

Skin diseases associated with inflammation or oxidative stress represent the most common problem in dermatology. The present study demonstrates that fish scale collagen peptides (FSCP) protect against CoCl2-induced cytotoxicity and TNF-α-induced inflammatory responses in human HaCaT keratinocyte cells. Our study is the first to report that FSCP increase cell viability and ameliorate oxidative injury in HaCaT cells through mechanisms mediated by the downregulation of key proinflammatory cytokines, namely, TNF-α, IL-1β, IL-8, and iNOS. FSCP also prevent cell apoptosis by repressing Bax expression, caspase-3 activity, and cytochrome c release and by upregulating Bcl-2 protein levels in CoCl2- or TNF-α-stimulated HaCaT cells. In addition, the inhibitory effects of FSCP on cytotoxicity and the induction of proinflammatory cytokine expression were found to be associated with suppression of the ROS, MAPK (p38/MAPK, ERK, and JNK), and NF-κB signaling pathways. Taken together, our data suggest that FSCP are useful as immunomodulatory agents in inflammatory or immune-mediated skin diseases. Furthermore, our results provide new insights into the potential therapeutic use of FSCP in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries.

Microscale Diffusion Measurements and Simulation of a Scaffold with a Permeable Strut

Electrospun nanofibrous structures provide good performance to scaffolds in tissue engineering. We measured the local diffusion coefficients of 3-kDa FITC-dextran in line patterns of electrospun nanofibrous structures fabricated by the direct-write electrospinning (DWES) technique using the fluorescence recovery after photobleaching (FRAP) method. No significant differences were detected between DWES line patterns fabricated with polymer supplied at flow rates of 0.1 and 0.5 mL/h. The oxygen diffusion coefficients of samples were estimated to be ~92%–94% of the oxygen diffusion coefficient in water based on the measured diffusion coefficient of 3-kDa FITC-dextran. We also simulated cell growth and distribution within spatially patterned scaffolds with struts consisting of either oxygen-permeable or non-permeable material. The permeable strut scaffolds exhibited enhanced cell growth. Saturated depths at which cells could grow to confluence were 15% deeper for the permeable strut scaffolds than for the non-permeable strut scaffold.

Fabrication of In Vitro Cancer Microtissue Array on Fibroblast-Layered Nanofibrous Membrane by Inkjet Printing

In general, a drug candidate is evaluated using 2D-cultured cancer cells followed by an animal model. Despite successful preclinical testing, however, most drugs that enter human clinical trials fail. The high failure rates are mainly caused by incompatibility between the responses of the current models and humans. Here, we fabricated a cancer microtissue array in a multi-well format that exhibits heterogeneous and batch-to-batch structure by continuous deposition of collagen-suspended Hela cells on a fibroblast-layered nanofibrous membrane via inkjet printing. Expression of both Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) was higher in cancer microtissues than in fibroblast-free microtissues. The fabricated microtissues were treated with an anticancer drug, and high drug resistance to doxorubicin occurred in cancer microtissues but not in fibroblast-free microtissues. These results introduce an inkjet printing fabrication method for cancer microtissue arrays, which can be used for various applications such as early drug screening and gradual 3D cancer studies.

Co-targeting of Tiam1/Rac1 and Notch ameliorates chemoresistance against doxorubicin in a biomimetic 3D lymphoma model

Lymphoma is a heterogeneous disease with a highly variable clinical course and prognosis. Improving the prognosis for patients with relapsed and treatment-resistant lymphoma remains challenging. Current in vitro drug testing models based on 2D cell culture lack natural tissue-like structural organization and result in disappointing clinical outcomes. The development of efficient drug testing models using 3D cell culture that more accurately reflects in vivo behaviors is vital. Our aim was to establish an in vitro 3D lymphoma model that can imitate the in vivo 3D lymphoma microenvironment. Using this model, we explored strategies to enhance chemosensitivity to doxorubicin, an important chemotherapeutic drug widely used for the treatment of hematological malignancies. Lymphoma cells grown in this model exhibited excellent biomimetic properties compared to conventional 2D culture including (1) enhanced chemotherapy resistance, (2) suppressed rate of apoptosis, (3) upregulated expression of drug resistance genes (MDR1, MRP1, BCRP and HIF-1α), (4) elevated levels of tumor aggressiveness factors including Notch (Notch-1, -2, -3, and -4) and its downstream molecules (Hes-1 and Hey-1), VEGF and MMPs (MMP-2 and MMP-9), and (5) enrichment of a lymphoma stem cell population. Tiam1, a potential biomarker of tumor progression, metastasis, and chemoresistance, was activated in our 3D lymphoma model. Remarkably, we identified two synergistic therapeutic oncotargets, Tiam1 and Notch, as a strategy to combat resistance against doxorubicin in EL4 T and A20 B lymphoma. Therefore, our data suggest that our 3D lymphoma model is a promising in vitro research platform for studying lymphoma biology and therapeutic approaches.