다양한 기질에서 골수 중간엽 줄기세포의 3차원 배양 및 생체이식에 의한 골형성능

이청송(Qing Song Li),윤택림(Taek Rim Yoon),김형근(Hyung Keun Kim),노윤정(Yoon Jung Roh),정은정(Eun Jung Jung) 대한정형외과학회 2006 대한정형외과학회지 Vol.41 No.6

목적: 다양한 지지체를 이용한 생체내 골형성과 최적의 세포배양 지지체를 알아보기 위해서 골아세포의 3차원 배양과 지지체들의 적합성을 비교하였다. 대상 및 방법: 가토골수에서 분리한 중간엽 줄기세포를 골아세포로 분화, 유도한 후 골형성 단백질의 발현과 석회화 양상을 관찰하였다. 또한 여러 지지체를 이용하여 3차원 배양하고 복합체를 주사현미경으로 관찰하였다. 복합체를 누드마우스에 이식하여 방사선, 조직관찰, 면역조직화학 등의 방법으로 골형성 여부를 확인하였다. 결과: 중간엽 줄기세포는 골아세포로 분화하였으며 석회화가 관찰되었다. 골형성 단백질의 발현이 2-3주 이후 지속적으로 관찰되었다. Osteocalcin 생성은 6-7주에서 급격히 생성되었다. 골아세포는 hydroxyapatite보다 사람뼈나 돼지뼈에서 많이 부착 증식하였고, PLGA에서는 세포가 밀착하지 않고 그물망 구조를 형성하였다. 칼슘알긴산염 지지체에서는 살아있는 세포를 관찰할 수 없었다. 그렇지만 생체이식한 사람뼈, 돼지뼈, hydroxyapatite, PLGA에서 풍부한 양의 세포와 신생조직을 관찰할 수 있었다. 결론: 다양한 합성 고분자보다는 천연골이 삼차원적 세포 배양 및 단기간의 동물실험에서 더 효과적이고 적합한 지지체임을 확인할 수 있었다. Purpose : To determine the in vivo bone formation on an osteoblastic cells-scaffold composite and the best scaffold for bone formation. This study focused on the three-dimensional culture of osteoblastic cells using various tissue-engineered scaffolds and compared the suitability of those scaffolds. Materials and Methods: Mesenchymal stem cells were harvested from New Zealand white rabbits and were differentiated into osteoblastic cells. The expression of osteogenic proteins in osteoblastic cells was observed by RT-PCR and a phase of mineralization was checked using von Kossa staining. In addition, three-dimensional cultured osteoblastic cells-scaffolds composites using a variable scaffold were observed by scanning electron microscopy. The cell-scaffold composites were transplanted into athymic nude mice. X-ray, a histochemical study, and immunohistochemistry were used to evaluate the in vivo bone formation. Results : The mesenchymal stem cells proliferated quite well in the early phase and differentiated into osteoblastic cells by an inducing substance. The osteoblastic cells were observed as spindle and polygonal shapes and were mineralized. Expression of the osteoblastic proteins was observed continuously after 2-3 weeks. The amount of osteocalcin secretion increased for 6-7 weeks. The osteoblastic cells adhered more to the human and porcine bone than to the hydroxyapatite-scaffold. In the case of the PLGA scaffold, the cells proliferated and formed a net-like structure but did not adhere well. No Living cells were observed in the calcium alginate scaffold. However, after the in vivo transplant, abundant cells and new tissue were observed within the human bone, porcine bone, hydroxyapatite, and PLGA but not within the calcium alginate scaffold. Conclusion : In the three-dimensional cultures, natural bone was found to be a more effective and suitable scaffold for cell culture than the various synthetic polymers.

3 차원 Blended PCL (60 wt %)/β-TCP (40 wt %) 인공지지체의 제작 및 특성 평가

사민우(Min-Woo Sa),김종영(Jong Young Kim) 대한기계학회 2014 大韓機械學會論文集A Vol.38 No.4

조직 공학에 있어 인공지지체는 손상된 조직 및 기관의 기능을 재생하기 위한 거푸집으로 제공되며 3 차원 구조물이다. 인공지지체의 재료 중에서 폴리카프로락톤(Polycaprolactone, PCL)과 삼인산칼슘(β-tricalcium phosphate, β-TCP)은 생분해성과 생체적합성을 가지고 있다. 본 연구에서는 다축 인공지지체 제작 시스템을 이용하여 3 차원 PCL, blended PCL(60 wt %)/β-TCP(40 wt %), 그리고 β-TCP 인공지지체를 제작하였다. 제작된 인공지지체는 주사전자현미경 분석을 통해 600 ± 20 μm 의 공극 크기로 잘 제작되었다. 기계적 특성 평가를 통해 3 차원 PCL, blended PCL(60 wt %)/β-TCP(40 wt %), 그리고 β-TCP 인공지지체의 효과는 분석되었다. 게다가 Saos-2 세포를 이용한 in vitro 연구를 수행하여 세포 증착 및 증식과 같은 세포 거동에 의한 3 차원 인공지지체의 효과를 확인하였다. 요컨대 3D blended PCL(60 wt %)/β-TCP(40 wt %)인공지지체가 압축 강도와 생체적합성 그리고 골전도성에 있어서 인체의 해면골에 더욱 적합하였다. 따라서 3D 인공지지체의 제작에 있어 PCL 과 β-TCP 를 혼합하는 것은 효과적인 골 재생을 위해 촉망되는 전략이 될 것이다. In tissue engineering, a scaffold is a three-dimensional(3D) structure that serves as a template for regeneration the functions of damaged tissues or organs. Among materials for scaffolds, polycaprolactone(PCL) and β-tricalcium phosphate(β-TCP) are biodegradable and biocompatible. In this study, we fabricated 3D PCL, blended PCL (60 wt %)/β-TCP (40 wt %), and pure β-TCP scaffolds by a multi-head scaffold fabrication system. Scaffolds with a pore size of 600 ± 20 μm was observed by scanning electron microscopy. The effects of 3D PCL, blended PCL (60 wt %)/β-TCP (40 wt %) and pure β-TCP scaffolds were analyzed by evaluating their mechanical characteristics. In addition, in an in-vitro study using osteoblast-like saos-2 cells, we confirmed the effects of 3D scaffolds on cellular behaviors such as cell adhesion and proliferation. In summary, the 3D blended PCL (60 wt %)/β-TCP (40 wt %) scaffold was found to be suitable for human cancellous bone in terms of its the compressive strength, biocompatibility, and osteoconductivity. Thus, blending PCL and β-TCP could be a promising approach for fabricating 3D scaffolds for effective bone regeneration.

조직공학용 지지체의 비파괴 분석을 위한 표면 탄성파 시뮬레이션 및 고속초음파 기반 측정법

윤철희(Chulhee Yun),남승윤(Seung Yun Nam) 대한기계학회 2017 大韓機械學會論文集B Vol.41 No.12

조직공학용 세포지지체의 물성은 생체적합성과 세포의 거동 및 기능에 큰 영향을 준다. 이러한 세포지지체의 기계적 특성을 측정하는 기존의 방법들은 대부분 지지체의 파괴적 과정을 거치게 되며 동일 지지체내의 물성변화를 장기간 계속적으로 측정할 수 없는 제한이 있었다. 하지만, 실제 피부 질병 진단 등에 사용되어 온 표면 탄성파 측정법을 이용하면 점탄성을 가지는 세포지지체의 비파괴적 물성측정이 가능하다. 본 연구에서는 세포지지체 물성 변화의 비파괴적인 측정을 위해 집속 초음파 및 초고속 영상법을 이용하였다. 또한, 실제 세포지지체와 유사한 기계적 특성을 가지는 다양한 물성의 젤라틴 기반 샘플 및 점탄성을 조절할 수 있는 알긴산-금나노입자 기반 세포지지체를 제작하여 표면 탄성파의 전파를 시뮬레이션하고 이를 측정치와 비교 분석하였다. The physical properties of tissue-engineered scaffolds are strongly related with biocompatibility as well as cell behaviors and functions. Conventional methods including rheological measurement have been mostly destructive and hardly available for continuous monitoring of the viscoelastic changes for different time points. In this study, surface wave measurement were used for non-destructive assessment of the viscoelastic properties of the tissueengineered scaffolds. Specifically, the propagation of surface waves was simulated using the K-wave toolbox in Matlab to optimize beamforming parameters. In addition, surface wave velocity and shear modulus were calculated in gelatin based tissue mimicking phantoms and phototuning alginate gels. These were then compared with reference values obtained with a rheometer. The surface wave measurement using ultrafast ultrasound imaging offers the possibility of a nondestructive and longitudinal assessment of tissue-engineered scaffolds.

바이오리액터 개발과 기계적 자극에 의한 중간엽 줄기세포의 영향에 관한 연구

주민진(Min Jin Joo),전흥재(Heoung-Jae Chun),정형진(Hyung Jin Jung),이창근(Chang Gun Lee),허동녕(Dong Nyoung Heo),권일근(Il Keun Kwon),문성환(Seong Hwan Moon) 대한기계학회 2010 大韓機械學會論文集A Vol.34 No.6

중간엽 줄기세포(MSCs)는 미분화 상태의 세포로써, 섬유아세포, 연골아세포, 골아세포 등으로 분화하여 인체의 근골격계를 구성하며, 기계적 자극은 중간엽 줄기세포 분화를 결정하는 중요한 인자로 알려져 이다. 본 연구에서는, 세포가 생존하기 위한 환경을 제공하고, 세포가 기계적 자극조건에 따라 분화할 수 있도록 하는 바이오리엑터를 제안하엿다. 또한, 중간엽 줄기세포를 배양하기 위한 세포 지지체로써 PU(polyurethane)로 제작된 지지체를 제안하였다. 세포 분화를 확인하기 위하여, 중간엽 줄기세포를 PU 지지체에 seeding한 후, 바이오리엑터를 이용하여 기계적 자극에 의한 세포의 분화를 확인하였다. It is well known that mesenchymal stem cell(MSCs) can be differentiated into fibroblasts, chondrocytes, and osteoblasts and that they develop into fibrous tissue, cartilage, or bone, as a result of mechanical stimulation. In this study, we developed a bioreactor system, which is composed of a reactor vessel that provides the required cell culture environment, an environment controlling chamber to control the media, a gas mixer, and a reactor motion control subsystem to apply mechanical stimuli to the cells. For the MSC culture, We used a poly-urethane (PU) scaffold, with a collagen coating to ensure improved cohesion ratio. Then, we transferred the cultivated MSCs in the PU scaffold, cultured the cells in the bioreactor system, and confirmed the proliferation, differentiation, and ossification processes, resulting from mechanical stimuli.

나노-마이크로 정밀 분사 시스템을 이용한 하이브리드 인공지지체의 제작 및 평가

하성우,김종영 대한기계학회 2014 大韓機械學會論文集A Vol.38 No.8

최근에, 3 차원 인공지지체와 나노섬유는 골 조직 재생을 위해 개발되고 있다. 본 연구에서는, 나노-마이크로 정밀 분사 시스템을 이용하여 하이브리드 인공지지체를 제작하였다. 하이브리드 인공지지체는 마이크로 인공지지체와 나노섬유가 결합하여 제작되었으며, 마이크로 인공지지체와 나노섬유를 얻기 위해 자유 형상 제작 기술과 전기방사 기법이 사용되었다. 마이크로 인공지지체는 정밀한 공극을 고려하여 CAD/CAM 데이터 따라 자유 형상 제작 기술에 의해 제작되었으며, 제작 공정은 100 °C 의 온도, 평균 650 kPa 의 압력, 그리고 250 mm/sec 의 Z 축 이송속도가 적용되었다. 그리고 전기방사법을 통하여 나노섬유를 제작함에 있어서 본 시스템에 적용한 공정 조건은, 5 kV 의 전압, 0.1 ml/min 의 유량, 그리고 1 mm 의 노즐 팁과 콜렉터와의 거리로 설정하였다. 제작된 하이브리드 인공지지체는 MG-63 세포를 이용하여 세포 증식 실험을 진행하였다. Recently, three-dimensional scaffolds and nanofibers are being developed for bone tissue regeneration. In this study, we fabricated a hybrid scaffold using a nano-micro precision deposition system. The fabrication process involved the application of the solid freeform fabrication (SFF) technology and electrospinning. The hybrid scaffolds were combined using micro scaffolds and nanofibers. The nanofibers were deposited on each layer of the micro scaffolding using the electrospinning process. The micro scaffolds were fabricated using the SFF technology at a temperature of 100 °C, pressure of 650 kPa, and scan velocity of 250 mm/s. Nanofiber fabrication was conducted by means of electrospinning using the flow rate, solution concentration, distance from the tip to the collector (TCD), and voltage. The nanofibers were fabricated using a flow rate of 0.1 ml/min, voltage of 5 kV, TCD of 1 mm, and 10 wt% of solution concentration. MG-63 cells were seeded into the hybrid scaffold for the purpose of its evaluation.

기계적 물성이 향상된 바이오프린팅 복합재-카고메 구조 세포지지체 제작

조용상(Yong Sang Cho) 대한기계학회 2020 大韓機械學會論文集B Vol.44 No.11

골 조직공학에서 기존의 골 이식재 한계를 극복하고자 생체 적합성/생분해성 합성 고분자 및 생체 적합성 바이오세라믹이 바이오프린팅 세포지지체 제작에 사용되고 있다. 하지만 합성 고분자의 낮은 생체 적합성, 바이오세라믹의 낮은 기계적 물성으로 인한 한계가 보고되었다. 따라서 본 연구에서는 골조직 재건용 바이오프린팅 세포지지체의 기계적 물성을 향상시키기 위해 생분해성 합성 고분자/바이오세라믹 복합재와 초경량 구조체인 카고메 구조를 갖는 바이오프린팅 세포지지체를 제안하였다. 게다가 격자 구조를 갖는 바이오프린팅 세포지지체를 이용하여 바이오프린팅 복합재-카고메 세포지지체의 구조적 특성, 기계적 물성, 생체 외 세포 배양 특성을 비교 평가하였다. In bone tissue engineering, to overcome the limitations of traditional bone grafts, biocompatible and biodegradable synthetic polymers and bioceramics are used to fabricate bioprinted scaffolds. However, synthetic polymers have relatively low biocompatibility, and bioceramics are brittle. Therefore, in this study, to enhance the mechanical properties of bioprinted scaffolds for bone regeneration, an ultra-light composite bioprinted scaffold consisting of biodegradable polymer and bioceramic materials with a Kagome structure was proposed. Moreover, the structural characteristics, mechanical properties, and in vitro cell response of the composite Kagome scaffold fabricated using the bioprinting system were assessed and compared with those of a bioprinted scaffold with a grid structure.

임의 형상 제작 기법을 이용한 3차원 세포지지체 제작에 관한 연구

최도현(Do-Hyun Choi),김현철(Hyun-Chul Kim) 한국기계가공학회 2019 한국기계가공학회지 Vol.18 No.2

With the goal of tissue regeneration for organs damaged through an accident or a disease, research on tissue engineering has been conducted to produce 3-D scaffolds that can support the cells in the attachment and growth for the cell proliferation and differentiation. A scaffold requires a suitable pore size and porosity to increase the nutrient circulation or oxygen supply for the attachment and growth of cells. The existing production methods such as solvent-casting particulate leaching, phase separation, and fiber bonding have certain disadvantages. With these methods, it is difficult to obtain a free desired shape. In addition, certain pore sizes and interconnectivities among the pores may not be guaranteed. To solve these problems, this study has fabricated a scaffold with a 3-D shaped nose using Alginate, which is a natural polymer obtained through Fused Deposition Modeling (FDM), one of the CAD/CAM-based Solid Freeform Fabrication (SFF) methods.

이중기공 세포지지체 제작을 위한 powder extruder 개발

강내운(Nae-Woon Kang),이세환(Se-Hwan Lee),김희경(Hee-Kyeong Kim),조영삼(Young-Sam Cho),이승재(Seung-Jae Lee) 대한기계학회 2015 대한기계학회 춘추학술대회 Vol.2015 No.11

Prior to our study, a novel technique was developed through combining SLUP(Salt Leaching Using Powder) and WNM(Wire-Network Molding) techniques to fabricate dual-pore scaffold. The advantages of this technique are solvent-free, high temperature-free, the pore interconnectivity and high porosity. Additionally, cell-proliferation(in vitro) of a dual-pore scaffold was faster than that of a scaffold fabricated by bio-plotting and SLUP technique. However these techniques have a disadvantage. For example, the scaffold fabrication process was manually. Therefore, it takes a long time and effort to make several scaffolds. In this study, we developed the powder extruder to fabricate dual-pore scaffold. The material was used for PCL(Polycaprolactone) and NaCl. The powder extruder was used as the basis for a 3D printer(Delta bot). The head of powder extruder was extruded using a screw. We compared characteristics of SLUP&WNM method with the powder extruder method for dual-pore scaffold fabrication

SLUP+WNM 세포지지체에서 osteoblast cell과 saos-2 cell의 in-vitro 세포 배양 특성 비교

조용상(Yong Sang Cho),홍명화(Myung Wha Hong),김소연(So-Youn Kim),이승재(Seung-Jae Lee),이준희(Jun Hee Lee),김영율(Young Yul Kim),조영삼(Young-Sam Cho) 대한기계학회 2013 대한기계학회 춘추학술대회 Vol.2013 No.12

In this study, a novel technique for dual-pore scaffold fabrication using SLUP (salt leaching using powder) and WNM (wire network molding) was proposed. To fabricated dual-pore scaffold, firstly, PCL with size of 63~100mm and NaCl powders with size of 100~180mm were prepared. Secondly, prepared PCL and NaCl powders were mixed using the stirrer at a certain ratio. Subsequently, stainless-steel needles with size of 500mm were inserted into a designed stainless steel mold. And then, the mixture powders were filled in the stainless-steel mold. Afterword, after the mold was pressurized at 30 MPa for 1 min using a lab-made compressor, the mold was heated to melt PCL powders in an oven at 100℃ for 20 min. Thereafter, after the stainless-steel needles were removed in the stainless-steel mold, the PCL/NaCl structure was separated from the stainless-steel mold. And then, the PCL/NaCl structure was soaked in D.I water for 24 hours using a sonicator to leach out the NaCl particles and dry for 24 hours in the desiccator. Consequently, the remaining PCL structure became the dual-pore scaffold. To analyze surface and cross-section characteristics of scaffolds, scaffolds was observed using SEM. Additionally, cell-culture experiments were performed using CCK-8 assay, ALP and compared with the bio-plotter and SLUP scaffolds.

마이크로 광 조형 기술로 제작된 3차원 인공지지체의 구조적 형태에 따른 연골세포의 생착 특성

이승재(S.-J. Lee),김병(B. Kim),임근배(G. Lim),김성원(S.-W. Kim),이종원(J.-w. Rhie),조동우(D.-W. Cho) 한국정밀공학회 2006 한국정밀공학회 학술발표대회 논문집 Vol.2006 No.5월

Understanding chondrocyte behavior inside complex, three-dimensional environments with controlled patterning of geometrical factors would provide significant insights into the basic biology of tissue regenerations. One of the fundamental limitations in studying such behavior has been the inability to fabricate controlled 3D structures. To overcome this problem, we have developed a three-dimensional microfabrication system. This system allows fabrication of predesigned internal architectures and pore size by stacking up the photopolymerized materials. Photopolymer SL5180 was used as the material for 3D scaffolds. The results demonstrate that controllable and reproducible inner-architecture can be fabricated. Chondrocytes harvested from human nasal septum were cultured in two kinds of 3D scaffolds to observe cell adhesion behavior. Such 3D scaffolds might provide effective key factors to study cell behavior in complex environments and could eventually lead to optimum design of scaffolds in various tissue regenerations such as cartilage, bone, etc. in a near future.