Development of Human Ear-mimetic Construct based on Three Dimensional Cell Printing Technology

Abstract

Tissue engineering is an interdisciplinary field integrating biotechnology, materials engineering and mechanical engineering that focuses on restoring and regenerating various tissues and organs, such as the bladder, airways, and myocardium. In particular, cell printing is a promising technology for effectively regenerating tissues and organs whereby a construct is fabricated based on a layer-by-layer process using appropriate cells at a high cell density, effective hydrogels, and growth factors. Such cell-printing technology allows three dimensional (3D) living tissues and organs to have anatomical cell arrangements and geometrical shapes similar to native tissues and organs by directly printing cells. Despite the outstanding potential of cell-printing technology, most studies remain in the early stages of regenerating tissues, with relatively simple shapes and functions. Furthermore, there is a limitation with regard to regenerating composite tissues of similar shape and size to human tissues and organs because the technology needed to fabricate complex-shaped constructs of large volumetric size has not yet been developed. In this research, a 3D human ear-mimetic cell-printing technology was developed and applied to ear regeneration. Here, the 3D human ear-mimetic cell-printing technology was validated through fabricating a human ear-mimetic cell-printed construct and evaluating the cartilage and adipose tissue formation. Human tissues and organs are composite tissues, comprising two or more types of cells and tissues. With respect to fabricating the cell-printed construct, a multi-head tissue/organ building system (MtoBS) with six independent dispensing heads was developed to enable the dispensing of widely varying biomaterials with high- and low-viscosity properties. Additionally, in fabricating the human ear-mimetic cell-printed construct, the prolonged 3D printing time can cause low cell viability and inadequate performance of the construct because the cells can be exposed to a harsh environment over a long printing time. With this in mind, the MtoBS was improved, with a clean-air workstation, a humidifier, and a Peltier system, providing a more suitable printing environment for a large-volume construct to maintain high cell viability. With the advanced MtoBS, it was confirmed that better control of the printing temperature enabled the enhanced printability of hydrogels and higher cell viability for the construct, despite a prolonged printing time. The human ear has a complex shape and an anatomically complex composition of tissues. A bottom-up fabrication method has the limitation of not being able to stack constructs with overhanging, curved, or hollowed shapes, given the cell-printing technology. To fabricate a cell-printed construct with a complex shape, a sacrificial layer process and computer-aided design and manufacturing (CAD/CAM) technologies were developed. In the sacrificial layer process, the main part was printed with poly-caprolactone (PCL) and the cell-laden hydrogel, and polyethylene glycol (PEG) was then deposited as a sacrificial layer to support the main structure. PEG can be removed readily in aqueous solutions, and the procedure for removing PEG does not affect cell viability. CAD/CAM software was developed to enable a cell-printed construct to be fabricated with two polymers and two cell-laden hydrogels by independent control of the dispensing heads. Fabrication conditions were established for creating a construct for ear regeneration. Appropriate line widths and pore sizes were determined for fabricating the construct with an ear-like shape and similar mechanical properties to those of the human ear through the evaluation of mechanical strength. Once the fabrication conditions were established, the sacrificial layer process, and cell-printing technologies allowed an ear-shaped cellular construct to be manufactured. According to these results, the advanced MtoBS enabled a cell-printed construct with complex shapes to be fabricated while maintaining high cell viability using the sacrificial layer process and CAD/CAM technology. For the effective regeneration of composite tissue in the ear, porcine auricular cartilage and human adipose tissue-derived decellularized extracellular (ear-cdECM and adECM) hydrogels were developed for printing cells and inducing target tissue formation. Human adipose-derived stem cells (ASC) were encapsulated with 2% ear-cdECM and 3% adECM hydrogels, and the cell-laden hydrogels were used to fabricate the cell-printed construct, which regenerated cartilage and adipose tissue in both in vitro and in vivo tests. Compared with control groups, which consisted of cell-printed constructs with 4% alginate hydrogel and transforming growth factor-beta (TGF‒β) for cartilage tissue formation, and with 3% collagen hydrogel and basic fibroblast growth factor (bFGF) for adipose tissue formation, it was confirmed that the two kinds of dECM hydrogels induced cartilage and adipose tissue formation at the same level of tissue regeneration as with specific growth factors. Based on the powerful dECM effect, the cell-printed construct in the shape of an ear was fabricated successfully with ASC-laden ear cdECM and adECM hydrogels, and was implanted subcutaneously in a nude mouse model for in vivo testing. For 4 and 8 weeks, the human ear-mimetic cell-printed constructs maintained their initial shapes, and cartilage and adipose tissue were formed in the parts with ear-cdECM and adECM hydrogels. These results demonstrated that dECM hydrogel can induce target tissue formation without specific growth factors and allow the cell-printed construct to form composite tissues. Thus, this validated the 3D cell-printing technology developed for fabricating human ear-mimetic cell printed constructs and regenerating composite tissues.