• 대한두개안면성형외과학회지
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• 제19권3호
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• pp.181-189
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• 2018

Background: Autogenous bone grafts have several limitations including donor-site problems and insufficient bone volume. To address these limitations, research on bone regeneration is being conducted actively. In this study, we investigate the effects of a three-dimensionally (3D) printed polycaprolactone (PCL)/tricalcium phosphate (TCP) scaffold on the osteogenic differentiation potential of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs). Methods: We investigated the extent of osteogenic differentiation on the first and tenth day and fourth week after cell culture. Cytotoxicity of the 3D printed $PCL/{\beta}-TCP$ scaffold was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, prior to osteogenic differentiation analysis. ADSCs and BMSCs were divided into three groups: C, only cultured cells; M, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold; D, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold with a bone differentiation medium. Alkaline phosphatase (ALP) activity assay, von Kossa staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed for comparative analysis. Results: ALP assay and von Kossa staining revealed that group M had higher levels of osteogenic differentiation compared to group C. RT-PCR showed that gene expression was higher in group M than in group C, indicating that, compared to group C, osteogenic differentiation was more extensive in group M. Expression levels of proteins involved in ossification were higher in group M, as per the Western blotting results. Conclusion: Osteogenic differentiation was increased in mesenchymal stromal cells (MSCs) cultured in the 3D printed PCL/TCP scaffold compared to the control group. Osteogenic differentiation activity of MSCs cultured in the 3D printed PCL/TCP scaffold was lower than that of cells cultured on the scaffold in bone differentiation medium. Collectively, these results indicate that the 3D printed PCL/TCP scaffold promoted osteogenic differentiation of MSCs and may be widely used for bone tissue engineering.

• 한국정밀공학회지
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• 제26권1호
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• pp.146-154
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• 2009

In recent tissue engineering field, it is being reported that the fabrication of 3D scaffolds having high porous and controlled internal/external architectures can give potential contributions in cell adhesion, proliferation and differentiation. To fabricate these scaffolds, various solid free-form fabrication technologies are being applied. The solid free-form fabrication technology has made it possible to fabricate solid free-form 3D microstructures in layer-by-layer manner. In this research, we developed a multi-head deposition system (MHDS) and used design of experiment (DOE) to fabricate 3D scaffold having an optimized internal/external shape, Through the organization of experimental approach using DOE, the fabrication process of scaffold, which is composed of blended poly-caprolactone (PCL), poly-lactic-co-glycolic acid (PLGA) and tricalcium phosphate (TCP), is established to get uniform line width, line height and porosity efficiently Moreover, the feasibility of application to the tissue engineering of MHDS is demonstrated by human bone marrow stromal cells (hBMSCs) proliferation test.

• Macromolecular Research
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• 제14권5호
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• pp.565-572
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• 2006

Recombinant human bone morphogenic protein-2 (rhBMP-2), which is known as one of the major local stimuli for osteogenic differentiation, was immobilized on the surface of hyaluronic acid (HA)-modified poly$(\varepsilon-caprolactone)$ (PCL) (HA-PCL) scaffolds to improve the attachment, proliferation, and differentiation of human bone marrow stem cells (hBMSCs) for bone tissue engineering. The rhBMP-2 proteins were directly immobilized onto the HA-modified PCL scaffolds by the chemical grafting the amine groups of proteins to carboxylic acid groups of HA. The amount of covalently bounded rhBMP-2 was measured to 1.6 pg/mg (rhBMP/HA-PCL scaffold) by using a sandwich enzyme-linked immunosorbant assay. The rhBMP-2 immobilized HA-modified-PCL scaffold exhibited the good colonization, by the newly differentiated osteoblasts, with a statistically significant increase of the rhBMP-2 release and alkaline phosphatase activity as compared with the control groups both PCL and HA-PCL scaffolds. We also found enhanced mineralization and elevated osteocalcin detection for the rhBMP-2 immobilized HA-PCL scaffolds, in vitro.

• Macromolecular Research
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• 제13권4호
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• pp.285-292
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• 2005

We developed alginate beads loaded with transforming growth $factor-{\beta}_{1}(TGF-{\beta}_{1})$ to examine the possible application of the scaffold and cytokine carrier in tissue engineering. In this study, bone marrow stromal cells (BMSCs) and $TGF{\beta}_{1}$ were uniformly encapsulated in the alginate beads and then cultured in vitro. The cell morphology and shape of the alginate beads were observed using inverted microscope, scanning electron microscope (SEM), histological staining and RT-PCR to confirm chondrogenic differentiation. The amount of the $TGF{\beta}_{1}$ released from the $TGF-{\beta}_{1}$ loaded alginate beads was analyzed for 28 days in vitro in a phosphate buffered saline (pH 7.4) at $37^{\circ}C$. We observed the release profile of $TGF-{\beta}_{1}$ from $TGF-{\beta}_{1}$ loaded alginate beads with a sustained release pattern for 35 days. Microscopic observation showed the open cell pore structure and abundant cells with a round morphology in the alginate beads. In addition, histology and RT-PCR results revealed the evidence of chondrogenic differentiation in the beads. In conclusion, these results confirmed that $TGF-{\beta}_{1}$ loaded alginate beads provide excellent conditions for chondrogenic differentiation.

• Journal of Biosystems Engineering
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• 제38권1호
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• pp.55-63
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• 2013

Purpose: The present study aimed at image processing methods for automatic cell pattern recognition and morphological analysis for tissue engineering applications. The primary aim was to ascertain the novel algorithm of adaptive brightness correction from microscopic images for use as a potential image analysis. Methods: General microscopic image of cells has a minor problem which the central area is brighter than edge-area because of the light source. This may affect serious problems to threshold process for cell-number counting or cell pattern recognition. In order to compensate the problem, we processed to find the central point of brightness and give less weight-value as the distance to centroid. Results: The results presented that microscopic images through the brightness correction were performed clearer than those without brightness compensation. And the classification of mixed cells was performed as well, which is expected to be completed with pattern recognition later. Beside each detection ratio of hBMSCs and HeLa cells was 95% and 92%, respectively. Conclusions: Using this novel algorithm of adaptive brightness correction could control the easier approach to cell pattern recognition and counting cell numbers.

• 폴리머
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• 제33권1호
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• pp.26-32
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• 2009

골 결손 치료를 위해 생분해성 고분자인 PLGA에 골다공증 치료제인 이프리플라본(IP)을 함유한 미립구를 O/W 유화 용매 증발법으로 제조하였으며, 생체외 방출실험에서 IP 방출량은 HPLC로 분석하였다. SEM을 이용하여 미립구에 부착된 세포의 거동을 확인하였으며, IP가 세포에 미치는 독성평가는 CCK-8 분석방법을 이용하여 측정하였다. 골 형성 지표인 ALP 활성도를 측정하였으며, 배양된 BMSCS의 골세포로의 표현형을 확인하기 위하여 RT-PCR을 수행하였다. 방출결과에서 IP 방출은 거의 40일 이상으로 지속적이었으며, IP를 함유한 미립구에서의 세포의 부착, 성장 등이 잘 이루어짐을 확인하였고, ALP 활성도 및 RT-PCR 분석결과에서도 단독의 PLGA 미립구보다 IP를 함유한 미립구에서의 값이 더 높은 것을 확인할 수 있었다. 본 실험결과를 바탕으로 향후 서방성 제제화에 있어서 IP/PLGA 미립구를 응용하게 되면 국소지향성 골분화 주사용 지지체로서 중요한 역할을 할 것으로 보인다.

• Molecules and Cells
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• 제40권2호
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• pp.123-132
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• 2017

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse ($Irs-1^{-/-}$) with growth retardation and subcutaneous adipocyte atrophy. $Irs-1^{-/-}$ mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of $Irs-1^{-/-}$ mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of $Irs-1^{-/-}$ mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

• 폴리머
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• 제33권2호
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• pp.104-110
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• 2009

탈미네랄화된 골분(DBP)은 생체활성 천연재료로서 골을 형성시키는 유도인자로 널리 사용되고 있다. 본 연구에서는 DBP 스폰지를 펩신의 농도와 가교제의 농도에 따라 그 특성을 분석함으로써 조직공학적 지지체로의 응용 가능성을 확인하였다. DBP를 3% 아세트산 용액에 48시간동안 용해시킬 시 0.1, 0.2 및 0.3% 펩신을 첨가하였고, 가교시 1, 5, 10, 50 및 100 mM의 EDC를 적용하였다. 이를 전자주사현미경, 시차주사열량계, 적외선 분광기, 다공도 및 흡수성 실험을 통해 특성분석하였으며 세포의 증식률은 MTT 분석으로 확인하였다. 이러한 결과를 통해 펩신의 농도로는 0.2$\sim$0.3%가, 가교제의 농도는 50$\sim$100 mM에서 물리화학적 특성이 우수한 것으로 판단되었고, 이러한 조건으로 제조된 DBP 스폰지는 조직 공학적 지지체로 유용하게 응용될 수 있을 것이다.