Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

The aim of this study was to develop novel polycaprolactone/poly(lactic-co-glycolic acid) (PCL/PLGA) scaffolds with a heparin-dopamine (Hep-DOPA) conjugate for controlled release of bone morphogenic protein-2 (BMP-2) to enhance osteoblast activity in vitro and also bone formation in vivo. PCL/PLGA scaffolds were prepared by a solid freeform fabrication method. The PCL/PLGA scaffolds were functionalized with Hep-DOPA and then BMP-2 was sequentially coated onto the Hep-DOPA/PCL/PLGA scaffolds. The characterization and surface elemental composition of all scaffolds were evaluated by scanning electron microscope and x-ray photoelectron spectroscopy. The osteoblast activities on all scaffolds were assessed by cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition in vitro. To demonstrate bone formation in vivo, plain radiograph, micro-computed tomography (micro-CT) evaluation and histological studies were performed after the implantation of all scaffolds on a rat femur defect. Hep-DOPA/PCL/PLGA had more controlled release of BMP-2, which was quantified by enzyme-linked immunosorbent assay, compared with Hep/PCL/PLGA. The in vitro results showed that osteoblast-like cells (MG-63 cells) grown on BMP-2/Hep-DOPA/PCL/PLGA had significantly enhanced ALP activity and calcium deposition compared with those on BMP-2/Hep/PCL/PLGA and PCL/PLGA. In addition, the plain radiograph, micro-CT evaluation and histological studies demonstrated that the implanted BMP-2/Hep-DOPA/PCL/PLGA on rat femur had more bone formation than BMP-2/Hep/PCL/PLGA and PCL/PLGA in vivo.

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Biophysical strain has been applied widely for bone regeneration. However, application of low-magnitude strains to cells on small-thickness scaffolds is problematic, especially in rodent calvarial defect models, because general translation systems have limitations in terms of generating low-magnitude smooth signals. To overcome these limitations, we developed an in vitro biophysical-stimulation platform for stimulation of cells on small-thickness scaffolds for rodent calvarial bone defects. The customized flexure-based translational nanoactuator enables generation of low-magnitude smooth signals at the subnano- to micrometer-scale. This nanoactuator, which is equipped with a piezoelectric actuator, is suitable for biological applications because it can generate friction-free motion with a high resolution. Moreover, its operation without wear or deterioration eliminates contamination factors in cell culture environments. The developed in vitro biophysical-stimulation platform using these nanoactuators showed predictable operational characteristics. Also, a few-micrometer sinusoidal signal was generated successfully without any distortion. Three-dimensional scaffolds fitting the critical-size rat calvarial defect model were fabricated using poly(caprolactone), poly(lactic-co-glycolic acid), and tricalcium phosphate. Runt-related transcription factor 2 expression was increased upon stimulation of human adipose-derived stem cells (ASCs) on these scaffolds were stimulated in the in vitro biophysical-stimulation platform. Additionally, the use of this platform resulted in up-regulation of alkaline phosphate, osteopontin, and osterix expression compared to the non-stimulated group. These preliminary in vitro results suggest that the biophysical environment provided by the in vitro biophysical-stimulation platform influences the osteogenic differentiation of ASCs.

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.