쾌속조형기반 3차원 세포 프린팅 기술 개발 및 복합 조직 재생으로의 적용

Abstract

This thesis presents a three-dimensional (3D) cell printing system including developments of a novel hybrid scaffold and new multi-head tissue/organ building system (MtoBS, one type of bioprinting apparatus) for regeneration of heterogeneous tissue.In the last few decades, many researchers have demonstrated the possibilities of tissue engineering by regenerating human tissues and organs such as skin, bladder, airway, and myocardium. Despite the remarkable potential of tissue engineering, the technique remains limited to thin tissues with simple structures or functions because the three essential components (scaffold, cell, and growth factor) of tissue engineering have not yet been harmoniously integrated into a desired form. Therefore, a revolutionary breakthrough for the appropriate fusion of the three main components will be required to overcome the current limitations in tissue engineering. In this respect, biofabrication aiming to invent novel biomedical products based on physical, chemical, biological, and/or engineering processes is regarded as a remarkable technology in tissue engineering. In particular, cell printing technology has been developed as a scaffold-free concept to overcome the limitations of scaffold-based tissue engineering. In particular, it allows 3D living tissues or organs to be manufactured directly based on a layer-by-layer deposition of cells with biologically relevant biomaterials. Moreover, the cell printing technology, which has the potential to produce versatile cell-laden structures, is able to mimic the anatomical cell arrangement of native tissues or organs. Thus far, however, the ideal 3D complex tissue or organ has not been achieved, as most printed structures have been produced using only hydrogel in the gel state, which exhibits unsuitable mechanical properties.To establish an effective 3D cell printing technology, a hybrid scaffold that is able to contain both synthetic biomaterials and natural hydrogel was newly fabricated using a multi-head deposition system (MHDS) previously developed in house. The hydrogel was intentionally infused into the space between the synthetic biomaterial fibers of the 3D scaffold. The in vitro cellular efficacy of the hybrid scaffold in proliferation and differentiation was evaluated using a mouse pre-osteoblast MC3T3-E1 cell line and human adipose derived stem cells. In addition, the collagen hydrogel encapsulating cells were dispensed into the synthetic biomaterial-based framework and the viability of the cells was assessed to investigate a possibility of hybrid scaffold for cell printing. In in vitro cellular assays, the hybrid scaffolds demonstrated superior effects on proliferation and differentiation of cells. Moreover, a high viability of the dispensed cells was measured.For a wide range of applications, the hybrid scaffold was supplemented with human recombinant bone morphogenetic protein-2 (rhBMP-2) to accelerate new bone formation in a rabbit radius segmental defect. rhBMP-2 is widely known to be a powerful growth factor facilitating active bone formation in both in vitro and in vivo applications. To control the release rate of rhBMP-2, collagen and gelatin hydrogel encapsulating rhBMP-2 was dispensed into hollow PCL/PLGA scaffolds. Long-term (up to 28 days) and short-term (within a week) rhBMP-2 delivery systems were achieved by PCL/PLGA/collagen and PCL/PLGA/gelatin hybrid scaffolds, respectively. An effective dose of 5μg/ml was determined by measuring the gene expression levels of osteogenic markers from human turbinate mesenchymal stromal cells (hTMSCs) seeded on the PCL/PLGA/collagen scaffold in vitro. In addition, it was verified that the burst release of rhBMP-2 from the PCL/PLGA/gelatin scaffold could not induce the osteogenic differentiation of hTMSCs in vitro at an equivalent dose. Results of in vivo animal experiments were consistent with those of in vitro studies. Micro-computed tomography (μCT) and histological analyses confirmed that sustained rhBMP-2 delivery systems showed the best bone healing quality at both weeks 4 and 8 after implantation. In contrast, a burst release of rhBMP-2 from the hybrid scaffold could hinder the bone healing process at an early stage due to the inflammatory response. In conclusion, the hybrid scaffold could be readily incorporated with bio-active growth factors to improve the regenerative capability of the scaffolds.In addition to the application to bone tissue engineering, the potential for cartilage regeneration of hybrid scaffolds encapsulating chondrocytes as well as transforming growth factor-β (TGF-β) were evaluated by in vitro and in vivo experiments. Alginate hydrogel encapsulating human chondrocytes with TGF-β was dispensed into every other pores of a PCL scaffold. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to identify glycosaminoglycans (GAGs), DNA, and total collagen content from different PCL/alginate hybrid scaffolds. The PCL/alginate hybrid scaffold containing TGF-β showed the highest extracellular matrix formation. The cell-laden hybrid scaffolds were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical (Alcian blue and hematoxylin and eosin (H&E) staining) and immunohistochemistry (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL/alginate/TGF-β hybrid scaffold. In conclusion, it was confirmed that three main components (scaffold, cells, and growth factor) could be effectively integrated into the hybrid scaffold using the MHDS. Moreover, it was demonstrated that the quality of regenerated cartilage could be improved by the comprehensive hybrid scaffold.According to the above results, a sophisticated manufacturing system enabling the use of suitable biomaterials in the proper locations, and in the exact amount was needed to improve the degree of tissue regeneration. Notably, in order to regenerate heterogeneous tissue consisting of two or more types of cells and tissues, the manufacturing system should be able to dispense a wide range of biomaterials having markedly different manufacturing conditions. In this respect, a multi-head tissue/organ building system (MtoBS) equipped with six dispensing heads was newly developed to produce 3D heterogeneous tissues or organs. The performance of the MtoBS was evaluated by measuring the line width of solid-state PCL and alginate solutions under various conditions. In addition, customized 3D porous structures for building osteochondral tissue (one of types of heterogeneous tissue), which had been previously attempted using the bioprinting technology with hydrogel only, were fabricated by sequential dispensing of PCL and two alginate solutions with and without two different types of live cells, osteoblasts and chondrocytes. The viability of the dispensed osteoblasts and chondrocytes was evaluated by live/dead cell assay.The in vivo feasibility of producing 3D biomimetic hybrid scaffolds by the MtoBS was investigated for osteochondral tissue regeneration in knee joint defects in rabbits. The bioprinting of two distinct layers consisting of different compositions has been successfully performed for the regeneration of osteochondral tissue using the MtoBS. Atelocollagen hydrogel encapsulating rhBMP-2 was dispensed for the subchondral bone regeneration. In particular, for the superficial cartilage regeneration, hyaluronic acid (HA) hydrogel gelated by host-guest interaction (host: cucurbituril[n] (n=5-8, 10

CB[n]), and guest: 1, 6-diaminohexane (DAH)) was used by encapsulating the TGF-β. It should be noted that no cytotoxic chemical reagents or physical external stimuli were included in the hydrogel formation process using the host-guest interaction of CB[6]/DAH and self-assembled fibril formation of collagen. Moreover, human turbinate mesenchymal stromal cells (hTMSCs), which were able to differentiate toward both chondrogenic and osteogenic lineages, were incorporated in both CB[6]/DAH-HA/TGF-β and collagen/rhBMP-2 [18-20]. To date, no studies have validated the outcomes of bioprinted 3D hybrid scaffolds in the osteochondral tissue regeneration using a knee joint defect of a rabbit. It was found that hybrid scaffolds comprised of distinct bio-mimetic compositions with natural ECM showed a superior potential for osteochondral tissue regeneration at week 8 in gross morphology examination and histological assays with H&E and MT.