3D cell printing of <i>in vitro</i> stabilized skin model and <i>in vivo</i> pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering

Kim, Byoung Soo,Kwon, Yang Woo,Kong, Jeong-Sik,Park, Gyu Tae,Gao, Ge,Han, Wonil,Kim, Moon-Bum,Lee, Hyungseok,Kim, Jae Ho,Cho, Dong-Woo Elsevier 2018 Biomaterials Vol.168 No.-

<P><B>Abstract</B></P> <P>3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during <I>in vitro</I> tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote <I>in vivo</I> wound healing. <I>In vivo</I> results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing.</P>

Intestinal Villi Model with Blood Capillaries Fabricated Using Collagen-Based Bioink and Dual-Cell-Printing Process

Kim, WonJin,Kim, GeunHyung American Chemical Society 2018 ACS APPLIED MATERIALS & INTERFACES Vol.10 No.48

<P>The human intestine, a vital organ in our digestive system, shows an anatomically complex architecture. The fabrication of three-dimensional (3D) intestinal models containing villus structures has been an important topic for intestine regeneration or organ-on-a-chip, because a 3D model can provide broad surface area and help absorption and transportation of digested nutrients. In this study, we developed a 3D intestinal villi model containing an epithelium layer and a blood capillary structure, using an innovative cell-printing process. The epithelium and capillary network of the 3D model were fabricated using two collagen-based bioinks laden with Caco-2 cells and human umbilical vein endothelial cells (HUVECs). The fabricating conditions were optimized to obtain a unique 3D villus structure, with capillary in the core and high cell viability. A fabricated single villus was 183 ± 12 μm in diameter and 770 ± 42 μm in height, which means the aspect ratio of the structure was 4.2 ± 0.3. The results indicate that the cell-laden intestinal villi successfully mimicked the 3D geometry of human intestinal villi. In vitro cellular activity of the 3D villi model containing epithelium and capillary demonstrated significantly higher cell growth and expression of enzymes and MUC17, compared to those of 2D models and a 3D villi model without the capillary network. The suggested 3D intestinal villi also exhibited the enhancement of the barrier function as compared to those of the others, and even demonstrated an increase of the permeability coefficient of FITC-dextran and glucose uptake ability (FITC, fluorescein isothiocyanate). These results indicate that a 3D intestinal villi model would be a highly promising for mimicking the human intestine.</P> [FIG OMISSION]</BR>

줄기세포 탑재 3차원 프린팅 polycarprolactone 스캐폴드

홍규식(Gyusik Hong),조정환(Jeong Hwan Cho),윤석환(Seokhwan Yun),최은정(Eunjeong Choi),안성민(Seongmin An),김정석(Jung Seok Kim),이재삼(Jae Sam Lee),심진형(Jin-hyung Shim),진송완(Songwan Jin),윤원수(Won-Soo Yun) 한국산학기술학회 2019 한국산학기술학회논문지 Vol.20 No.8

줄기세포를 기반으로 한 세포치료제는 생체 이식시 생착률이 낮아서 치료효과를 기대하기 어렵다. 이를 극복하기 위하여 줄기세포를 탑재할 수 있는 다양한 세포담체들이 개발되어 활용되고 있다. 이렇게 개발된 세포담체를 3-dimentional (3D) 프린팅하여 스캐폴드를 만들 경우, 환자의 손상부위 맞춤형 이식재를 제작할 수 있을 뿐만 아니라, 줄기세포를 탑재하여 손상부위를 기계적으로 보완하는 동시에 세포치료제로서의 효과도 얻을 수 있다. Polycaprolactone (PCL)은 저렴할 뿐 아니라 현재 가장 널리 쓰이고 있는 3D 프린팅 소재이기 때문에, PCL을 프린팅하여 세포담체로 활용할 경우 빠르고 경제적인 기술발전을 도모할 수 있다. 하지만 PCL 소재는 세포담체로서의 성능이 우수하지 못하여, 극히 일부의 세포만이 PCL 표면에서 생존한다. 본 연구에서는 이를 극복하기 위해서 PCL 소재에 세포의 탑재능력을 극대화되는 조건을 찾고자 하였다. PCL의 표면에 플라즈마를 처리하는 조건, PCL 표면을 콜라겐 코팅처리, PCL의 3D 프린팅 형상, 세포배양방법 변경 등 다양한 조건을 바탕으로 하여 PCL 소재에 인간 중간엽줄기세포의 세포탑재능력을 확인하였다. 세포탑재능력을 향상시킨다고 알려진 콜라겐 코팅과 플라즈마 처리를 적용하여, 플라즈마 처리 후 3% 콜라겐 코팅을 하였을 때 세포탑재능력이 가장 우수함을 확인하였고, 세포탑재능력에 영향을 줄 수 있는 세포배양방법과 스캐폴드의 구조변화를 적용하여, spheroid 세포배양시 기존의 단일세포배양법보다 탑재능력이 우수함을 확인하였으며, 스캐폴드의 구조는 세포탑재능력에 영향을 주지 못함을 확인하였다. 이를 바탕으로 PCL 소재를 세포담체로 활용한 다양한 연구를 시도하고자 한다. Stem cell therapy is not expected to bestow any therapeutic benefit because of the low engraftment rates after transplantation. Various cell-carrying scaffolds have been developed in order to overcome this problem. When the scaffold is formed by 3-dimensional (3D) printing, it is possible to create various shapes of scaffolds for specific regions of injury. At the same time, scaffolds provide stem cells as therapeutic-agents and mechanically support an injured region. PCL is not only cost effective, but it is also a widely used material for 3D printing. Therefore, rapid and economical technology development can be achieved when PCL is printed and used as a cell carrier. Yet PCL materials do not perform well as cell carriers, and only a few cells survive on the PCL surface. In this study, we tried to determine the conditions that maximize the cell-loading capacity on the PCL surface to overcome this issue. By applying a plasma treated condition and then collagen coating known to improve the cell loading capacity, it was confirmed that the 3% collagen coating after plasma treatment showed the best cell engraftment capacity during 72 hours after cell loading. By applying the spheroid cell culture method and scaffold structure change, which can affect the cell loading ability, the spheroid cell culture methods vastly improved cell engraftment, and the scaffold structure did not affect the cell engraftment properties. We will conduct further experiments using PCL material as a cell carrier and as based the excellent results of this study.

외이 재생을 위한 세포 프린팅 기반 복합 조직 구조물의 개발

이정섭(Jung-Seob Lee),정진우(Jin Woo Jung),심진형(Jin-Hyung Shim),오정훈(Jeong-Hoon Oh),조동우(Dong-Woo Cho) 대한기계학회 2013 대한기계학회 춘추학술대회 Vol.2013 No.12

Tissue engineering is an interdisciplinary field to regenerate and reconstruct damaged tissues and organs. Especially, the cell printing enabling the regeneration of the target tissue has been considered to be a promising technology. However, the cell printing technology is known to be difficult to fabricate the 3D composite tissue with complex shape. In this study, we used 3D printing technology including multiple cells positioning technology and a sacrificial layer process to regenerate various tissues as well as an ear. The main part was printed with poly-caprolactone (PCL) and multiple cells-laden hydrogel. At the same time, poly-ethylene-glycol (PEG) was also deposited as a sacrificial layer to support the main part. After complete fabrication, PEG can be easily removed in aqueous solutions, and the procedure for removing PEG has no effect on the cell viability. Though 3D printing technology, the various composite tissues were fabricated with the desired shape and separately printed cells in the structure also had good cell viability. As a result, the possibility of fabricating the complex composite tissue using 3D printing technology which allowed tissue formation was confirmed.

Electromagnetic field-assisted cell-laden 3D printed poloxamer-407 hydrogel for enhanced osteogenesis

( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2

3D bioprinted hydrogel has gained enormous attention, especially in tissue engineering, owing to its attractive structure and excellent biocompatibility. In this study, we demonstrated that 3D bioprinted cell-laden ‘thermoresponsive’ poloxamer-407 (P407) gels have the potential to stimulate osteogenic differentiation of apical papilla stem cells (SCAPs) under the influence of low voltage- frequency 1V-1Hz (0.14 mT), 5V-1Hz (0.62 mT), and 10V-1Hz (1.21 mT) electromagnetic fields (EMFs). Their exposure time was 5 min/day, 10 min/day, 20 min/day, and 30 min/day for each hydrogel group, respectively. The developed hydrogel exhibited higher mechanical strength as well as good printability, showing high-quality micro-architecture. Moreover, the as-printed hydrogels (5 mm × 5 mm) were loaded with plasminogen activator inhibitor-1 (PAI-1) for testing the combined effect of PAI-1 and EMFs on SCAP differentiation. Interestingly, the 3D hydrogels showed improved viability and differentiation of SCAPs under EMFs' influence as examined by live/dead assay and alizarin Red-S staining, respectively. Our results demonstrated that DSPP and DMP-1 markers' expression significantly increased (3.8-fold) in 5V (0.62 mT) EMFs treatment. A similar fashion was also observed in ALP and Col-1(2.80-fold), comparable to the control groups. Therefore, the higher expression of theses gene markers (DSPP, DMP-1 ALP, and Col-1) indicated their better osteogenic efficiency of P407-encapsulated SCAPs in the presence of EMFs. The results confirmed that P407 hydrogels are non-toxic for encapsulation of SCAPs, yielding high cell viability and accelerate the cell migration potential. The 3D hydrogels with PAI-1 exhibited high mRNA expression levels for osteogenic/odontogenic gene markers (ALP, Col-1, DSPP, and DMP-1) vis-à-vis control after 14 days of in vitro culture. Our findings suggest that 3D bioprinted P407 hydrogels are biocompatible for SCAP encapsulation, and the applied low voltage-frequency EMFs could effectively improve dental tissue regeneration, particularly for oral applications.

A 3D cell printed muscle construct with tissue-derived bioink for the treatment of volumetric muscle loss

Choi, Yeong-Jin,Jun, Young-Joon,Kim, Dong Yeon,Yi, Hee-Gyeong,Chae, Su-Hun,Kang, Junsu,Lee, Juyong,Gao, Ge,Kong, Jeong-Sik,Jang, Jinah,Chung, Wan Kyun,Rhie, Jong-Won,Cho, Dong-Woo Elsevier 2019 Biomaterials Vol.206 No.-

<P><B>Abstract</B></P> <P>Volumetric muscle loss (VML) is an irrecoverable injury associated with muscle loss greater than 20%. Although hydrogel-based 3D engineered muscles and the decellularized extracellular matrix (dECM) have been considered for VML treatment, they have shown limited efficacy. We established a novel VML treatment with dECM bioink using 3D cell printing technology. Volumetric muscle constructs composed of cell-laden dECM bioinks were generated with a granule-based printing reservoir. The 3D cell printed muscle constructs exhibited high cell viability without generating hypoxia and enhanced <I>de novo</I> muscle formation in a VML rat model. To improve functional recovery, prevascularized muscle constructs that mimic the hierarchical architecture of vascularized muscles were fabricated through coaxial nozzle printing with muscle and vascular dECM bioinks. Spatially printing tissue-specific dECM bioinks offers organized microenvironmental cues for the differentiation of each cell and improves vascularization, innervation, and functional recovery. Our present results suggest that a 3D cell printing and tissue-derived bioink-based approach could effectively generate biomimetic engineered muscles to improve the treatment of VML injuries.</P>

Recent Activities of Solid Oxide Fuel Cell Research in the 3D Printing Processes

주바이르 마사우드,무하마드 주바이르 칸,암자드 후세인,하피즈 아흐마드 이시팍,송락현,이승복,조동우,임탁형 한국수소및신에너지학회 2021 한국수소 및 신에너지학회논문집 Vol.32 No.1

Solid oxide fuel cell (SOFC) has received significant attention recently because of its potential for the clean and efficient power generation. The current manufacturing processes for the SOFC components are somehow complex and expensive, therefore, new and innovative techniques are necessary to provide a great deal of cell performance and fabricability. Three-dimensional (3D) printing processes have the potential to provide a solution to all these problems. This study reviews the literature for manufacturing the SOFC components using 3D printing processes. The technical aspects for fabrication of SOFC components, 3D printing processes optimization and material characterizations are discussed. Comparison of the SOFC components fabricated by 3D printing to those manufactured by conventional ceramic processes is highlighted. Further advancements in the 3D printing of the SOFC components can be a step closer to the cost reduction and commercialization of this technology.

3차원 세포 프린팅과 탈세포화 된 췌장 유래 세포외기질 바이오잉크를 이용한 3차원 췌장 전조직 구조체의 제작

김석원(Seok-Won Kim),장진아(Jinah Jang),한원일(Won-il Han),김송철(Song-Cheol Kim),조동우(Dong-Woo Cho) 대한기계학회 2015 대한기계학회 춘추학술대회 Vol.2015 No.11

Islet transplantation into the portal vein has been widely used for the treatment of type 1 diabetes. However, intra-hepatic transplantation has many disadvantages including lack of vasculature, low efficacy and gradual graft attrition. Bio-engineered pancreas potentially provide an alternative transplantation site such as subcutaneous or directly into pancreas for islets with improving blood supply and cell viability compared to conventional intra portal transplantation. In this study, we developed printable pancreas derived decelluarized extracellular matrix (pdECM) bioink which enhances islet functionality and long-term survival by mimicking pancreas microenvironment. pdECM bioink have been tested for INS1β-cell viability and proliferation. To fabricate the optimal pancreas tissue construct by 3D cell printing, we checked the proper cell concentration by encapsulating INS1β-cell into pdECM bioink in different concentration. Our result shows the biological utility of pdECM bioink as a suitable biomaterial to fabricate the 3D pancreas tissue construct and to enhance cell functionality.

Hydrogels with an embossed surface: An all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids

Kim, Se-jeong,Park, Jaesung,Byun, Hayeon,Park, Young-Woo,Major, Luke G.,Lee, Dong Yun,Choi, Yu Suk,Shin, Heungsoo Elsevier 2019 Biomaterials Vol.188 No.-

<P><B>Abstract</B></P> <P>Stem cell spheroids have been studied extensively in organoid culture and therapeutic transplantation. Herein, hydrogels with an embossed surface (HES) were developed as an all-in-one platform that can enable the rapid formation and culture of a large quantity of size-controllable stem cell spheroids. The embossed structure on the hydrogel was adjustable according to the grit designation of the sandpaper. Human adipose-derived stem cells (hADSCs) were rapidly assembled into spheroids on the hydrogel, with their size distribution precisely controlled from 95 ± 6 μm to 181 ± 15 μm depending on surface roughness. The hADSC spheroids prepared from the HES demonstrated expression of stemness markers and differentiation capacity. In addition, HES-based spheroids showed significantly greater VEGF secretion than spheroids grown on a commercially available low-attachment culture plate. Exploiting those advantages, the HES-based spheroids were used for 3D bioprinting, and the spheroids within the 3D-printed construct showed improved retention and VEGF secretion compared to the same 3D structure containing single cell suspension. Collectively, HES would offer a useful platform for mass fabrication and culture of stem cell spheroids with controlled sizes for a variety of biomedical applications.</P>

3D printing of a conductive polypyrrole-grafted gelatin methacrylate (GelMA)-based hydrogels for continuous microcurrent stimulation of human mesenchymal stem cells

( Sayan Deb Dutta ),( Keya Ganguly ),( Tejal Patel ),( Ki-taek Lim ) 한국농업기계학회 2021 한국농업기계학회 학술발표논문집 Vol.26 No.2

Electrical stimulation has been shown to ameliorate bone healing for a long time. This study developed a hybrid and 3D printable conductive methacrylated gelatin-polypyrrole (GelMA-PPy)-based photocurable and self-healing hydrogel inks for continuous microcurrent stimulation. For this, a custom-made electrical stimulation device (DC stimulation) was used to evaluate the osteogenic differentiation of human bone mesenchymal stem cells (hMSCs). The pyrrole was chemically grafted onto the surface of GelMA via a one-step conjugation reaction with ammonium persulfate (APS) and cross-linked with iron (III) chloride. The fabricated hydrogel was characterized by proton nuclear magnetic resonance (1H-NMR), scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) to analyze the grafting and chemical interaction. The as-prepared hydrogel exhibited higher mechanical and swelling properties compared to pure GelMA scaffolds. The 3D printed GelMA-PPy hydrogel showed improved cell viability (~12.6%) when exposed to 500 mV/day current, compared to pure GelMA after 7 and 14 days of cell culture. Interestingly,16.0~20.0-fold higher expression of osteogenic genes and protein markers (Runx2, ALP, OCN, and OPN) were observed in the GelMA-PPy treated groups than control suggest that pyrrole incorporation into GelMA matrix significantly improved the conductivity and osteogenic differentiation of hMSCs. Therefore, this study shows that the hMSCs react differentially to low-voltage DC electrical stimulation in the presence of GelMA-PPy scaffolds, which could be used as an ideal material for electrical stimulation for tissue engineering.