Engineered ECM-like microenvironment with fibrous particles for guiding 3D-encapsulated hMSC behaviours

Shin, Young Min,Kim, Taek Gyoung,Park, Jong-Seok,Gwon, Hui-Jeong,Jeong, Sung In,Shin, Heungsoo,Kim, Kyung-Soo,Kim, Dongyoon,Yoon, Myung-Han,Lim, Youn-Mook The Royal Society of Chemistry 2015 Journal of Materials Chemistry B Vol.3 No.13

<P>The alginate hydrogel has been used as an attractive scaffold for tissue regeneration. In particular, its simple cross-linking, high water absorption, and biocompatibility have facilitated its utility in regulating the interaction with cells or organs. However, three-dimensional (3D) networks of the alginate hydrogel do not provide fibrous anchorage sites such as the collagen fibres in the natural extracellular matrix (ECM). This has partially limited the survival of anchorage-dependent cells in the 3D hydrogel environment. In this report, we established a hybrid hydrogel containing fibrous particles (FP) that closely mimics the ECM. The RGD peptide-coupled FP (R-FP) has a wide range of distribution and was homogeneously dispersed in the hydrogel. The encapsulated human mesenchymal stem cells in the hydrogel could bind to the R-FP presenting remarkable spreading morphology, augmented viability and differentiation. These findings may elicit the significance of a physical interaction in which the R-FP provides structural and biological cues to the cells. This strategy can be widely applicable to a variety of hydrogel systems.</P>

Engineering Multi-cellular Spheroid Using Bioinspired Materials for Tissue Regeneration

Heungsoo SHIN 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.9

Engineering an aligned endothelial monolayer on a topologically modified nanofibrous platform with a micropatterned structure produced by femtosecond laser ablation

Shin, Young Min,Shin, Hyeok Jun,Heo, Yunhoe,Jun, Indong,Chung, Yong-Woo,Kim, Kyeongsoo,Lim, Youn Mook,Jeon, Hojeong,Shin, Heungsoo The Royal Society of Chemistry 2017 Journal of Materials Chemistry B Vol.5 No.2

<P>A monolayer of endothelial cells (ECs) aligned along the direction of blood flow plays crucial roles in the regulation of anti-thrombogenic and pro-inflammatory reactions in the blood vessel wall. Thus, many researchers have attempted to mimic the aligned structure of ECs in vascular grafts or tissue-engineered blood vessels. In the present study, we fabricated micro-groove patterned nanofibers using a femtosecond laser ablation technique to recapitulate the densely organized anisotropic architecture of the endothelial layer. Femtosecond laser ablation enabled us to generate high-resolution groove patterns (10 μm width) with 20 or 80 μm gaps on randomly oriented electrospun nanofibers. The patterned nanofibers exhibited anisotropic (transverse: 101.1 ± 4.0° and longitudinal: 123.5 ± 9.4°) water contact angles; however, the mechanical properties were consistent in both directions. The micropatterned nanofibers modulated the aligned structure or aspect ratio (20 μm: 0.23 ± 0.11 and 80 μm: 0.42 ± 0.18) of ECs along the pattern direction. In particular, the engineered aligned endothelial layer was effective in eliciting an anti-inflammatory response (approximately 50% greater than that of random or aligned nanofibers), thereby effectively preventing monocyte adhesion following activation by TNF-α treatment. Therefore, micropatterning by laser ablation can be utilized to generate high-resolution microgrooves on various substrates, thereby providing fundamental platforms for vascular tissue engineering.</P>

Advanced capability of radially aligned fibrous scaffolds coated with polydopamine for guiding directional migration of human mesenchymal stem cells

Shin, Young Min,Shin, Hyeok Jun,Yang, Dae-Hyeok,Koh, Young-Joo,Shin, Heungsoo,Chun, Heung Jae Royal Society of Chemistry 2017 Journal of Materials Chemistry B Vol. No.

<P>In a large tissue defect, faster migration of adjacent tissue toward the defect shortens the tissue regeneration time. Little has been explored on guiding of directional migration from all fronts of the defect boundary towards the center in tissue engineering. This paper demonstrates the effect of radially aligned fibrous scaffolds (RAFSs) coated with polydopamine in order to guide directional migration of human mesenchymal stem cells (hMSCs). RAFSs were electrospun using a collector with a set of electrodes, each constructed with a metallic ring and a point. The polydopamine was then coated by dipping the scaffolds in a dopamine solution (PD-RAFS). The RAFSs exhibited radial distribution of the fibers from the peripheral region toward the center, and polydopamine was uniformly coated over the entire surface by presenting characteristics of the aromatic ring from dopamine. When hMSCs were seeded on the scaffolds, cells grew in an elongated form toward the center along the fiber direction. In particular, the polydopamine coating improved adhesion and spreading of hMSCs on the scaffolds while preserving initial cell orientation. The hMSCs migrated toward the center of the scaffolds at the border of the seeded area; it was enhanced in the order of PD-RAFS > RAFS > random fibrous scaffolds. Therefore, PD-RAFSs can be utilized as an alternate scaffold that can lead to fast and directional migration of cells for finally facilitating tissue regeneration.</P>

Cell-interactive scaffolds for tissue engineering

Heungsoo SHIN 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.4

Mussel-Inspired Immobilization of Vascular Endothelial Growth Factor (VEGF) for Enhanced Endothelialization of Vascular Grafts

Shin, Young Min,Lee, Yu Bin,Kim, Seok Joo,Kang, Jae Kyeong,Park, Jong-Chul,Jang, Wonhee,Shin, Heungsoo American Chemical Society 2012 Biomacromolecules Vol.13 No.7

<P>Most polymeric vascular prosthetic materials have low patency rate for replacement of small diameter vessels (<5 mm), mainly due to failure to generate healthy endothelium. In this study, we present polydopamine-mediated immobilization of growth factors on the surface of polymeric materials as a versatile tool to modify surface characteristics of vascular grafts potentially for accelerated endothelialization. Polydopamine was deposited on the surface of biocompatible poly(<SMALL>l</SMALL>-lactide-<I>co</I>-ε-caprolactone) (PLCL) elastomer, on which vascular endothelial growth factor (VEGF) was subsequently immobilized by simple dipping. Surface characteristics and composition were investigated by using scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Immobilization of VEGF on the polydopamine-deposited PLCL films was effective (19.8 ± 0.4 and 197.4 ± 19.7 ng/cm<SUP>2</SUP> for DPv20 and DPv200 films, respectively), and biotin-mediated labeling of immobilized VEGF revealed that the fluorescence intensity increased as a function of the concentration of VEGF solution. The effect of VEGF on adhesion of HUVECs was marginal, which may have been masked by polydopamine layer that also enhanced cell adhesion. However, VEGF-immobilized substrate significantly enhanced proliferation of HUVECs for over 7 days of in vitro culture and also improved their migration. In addition, immobilized VEGF supported robust cell to cell interactions with strong expression of CD 31 marker. The same process was effective for immobilization of basic fibroblast growth factor, demonstrating the robustness of polydopamine layer for secondary ligation of growth factors as a simple and novel surface modification strategy for vascular graft materials.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bomaf6/2012/bomaf6.2012.13.issue-7/bm300194b/production/images/medium/bm-2012-00194b_0010.gif'></P>

세포흡착 거동의 기계적/생화학적 분석

신흥수(Heungsoo Shin) 대한기계학회 2008 대한기계학회 춘추학술대회 Vol.2008 No.11

Cell adhesion is a coordinated process involving initial binding of integrin receptors to extracellular matrix (ECM), recruitment of adhesion proteins, and focal adhesion assembly. The formation of mechanically stable focal adhesion assembly of cells within surrounding ECM is a key parameter to direct numerous cellular functions including cell migration, differentiation, and apotosis. With current cell adhesion assays, it is difficult to understand contributions of each coordinated event on evolution of cell adhesion strengthening since cells spontaneously spread upon their adhesion to the substrate, thus remodeling their cytoskeletal structure. In this presentation, novel approaches for analysis of cell adhesion strengthening process based on the combination of mechanical device, micro-patterned substrates, and molecular biological techniques will be discussed.

Fabrication of <i>in vitro</i> 3D mineralized tissue by fusion of composite spheroids incorporating biomineral-coated nanofibers and human adipose-derived stem cells

Ahmad, Taufiq,Shin, Hyeok Jun,Lee, Jinkyu,Shin, Young Min,Perikamana, Sajeesh Kumar Madhurakat,Park, So Yeon,Jung, Hyun Suk,Shin, Heungsoo Elsevier 2018 ACTA BIOMATERIALIA Vol.74 No.-

<P><B>Abstract</B></P> <P>Development of a bone-like 3D microenvironment with stem cells has always been intriguing in bone tissue engineering. In this study, we fabricated composite spheroids by combining functionalized fibers and human adipose-derived stem cells (hADSCs), which were fused to form a 3D mineralized tissue construct. We prepared fragmented poly (ι-lactic acid) (PLLA) fibers approximately 100 μm long by partial aminolysis of electrospun fibrous mesh. PLLA fibers were then biomineralized with various concentrations of NaHCO<SUB>3</SUB> (0.005, 0.01, and 0.04 M) to form mineralized fragmented fibers (mFF1, mFF2, and mFF3, respectively). SEM analysis showed that the minerals in mFF2 and mFF3 completely covered the fiber surface, and surface chemistry analysis confirmed the presence of hydroxyapatite peaks. Additionally, mFFs formed composite spheroids with hADSCs, demonstrating that the cells were strongly attached to mFFs and homogeneously distributed throughout the spheroid. <I>In vitro</I> culture of spheroids in the media without osteogenic supplements showed significantly enhanced expression of osteogenic genes including Runx2 (20.83 ± 2.83 and 22.36 ± 2.18 fold increase), OPN (14.24 ± 1.71 and 15.076 ± 1.38 fold increase), and OCN (4.36 ± 0.41 and 5.63 ± 0.51 fold increase) in mFF2 and mFF3, respectively, compared to the no mineral fiber group. In addition, mineral contents were significantly increased at day 7. Blocking the biomineral-mediated signaling by PSB 603 significantly down regulated the expression of these genes in mFF3 at day 7. Finally, we fused composite spheroids to form a mineralized 3D tissue construct, which maintained the viability of cells and showed pervasively distributed minerals within the structure. Our composite spheroids could be used as an alternative platform for the development of <I>in vitro</I> bone models, <I>in vivo</I> cell carriers, and as building blocks for bioprinting 3D bone tissue.</P> <P><B>Statement of Significance</B></P> <P>This manuscript described our recent work for the preparation of biomimeral-coated fibers that can be assembled with mesenchymal stem cells and provide bone-like environment for directed control over osteogenic differentiation. Biomineral coating onto synthetic, biodegradable single fibers was successfully carried out using multiple steps, combination of template protein coating inspired from mussel adhesion and charge-charge interactions between template proteins and mineral ions. The biomineral-coated single micro-scale fibers (1–2.5 μm in diameter) were then assembled with human adipose tissue derived stem cells (hADSCs). The assembled structure exhibited spheroidal architecture with few hundred micrometers. hADSCs within the spheroids were differentiated into osteogenic lineage <I>in vitro</I> and mineralized in the growth media. These spheroids were fused to form <I>in vitro</I> 3D mineralized tissue with larger size.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Agglomeration of human dermal fibroblasts with ECM mimicking nano-fragments and their effects on proliferation and cell/ECM interactions

Ahmad, Taufiq,Shin, Young Min,Lee, Jinkyu,Shin, Hyeok Jun,Madhurakart Perikamana, Sajeesh Kumar,Shin, Heungsoo THE KOREAN SOCIETY OF INDUSTRIAL AND ENGINEERING 2018 JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY -S Vol.67 No.-

<P><B>Abstract</B></P> <P>Here, we engineered spheroids by using ECM mimicking nano-fragments (NFs) with fibroblasts and investigated their effect on proliferation and cell/ECM interactions. NF incorporation resulted in formation of a stable spheroid, which improved proliferation and viability of cells by assisting oxygen transport confirmed by LOX-1 staining. In addition, hypoxic and apoptotic genes were significantly downregulated in spheroids with PD-NFs. Furthermore, ECM and cell junction proteins were highly expressed. Overall, our findings suggest that incorporation of NFs within spheroids for assembly with various cell types can be an alternative approach for 3D cell culture in many applications.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Hybrid-spheroids incorporating ECM like engineered fragmented fibers potentiate stem cell function by improved cell/cell and cell/ECM interactions

Ahmad, Taufiq,Lee, Jinkyu,Shin, Young Min,Shin, Hyeok Jun,Madhurakat Perikamana, Sajeesh Kumar,Park, Sun Hwa,Kim, Sung Won,Shin, Heungsoo Elsevier 2017 ACTA BIOMATERIALIA Vol.64 No.-

<P><B>Abstract</B></P> <P>Extracellular matrix (ECM) microenvironment is critical for the viability, stemness, and differentiation of stem cells. In this study, we developed hybrid-spheroids of human turbinate mesenchymal stem cells (hTMSCs) by using extracellular matrix (ECM) mimicking fragmented fibers (FFs) for improvement of the viability and functions of hTMSCs. We prepared FFs with average size of 68.26 µm by partial aminolysis of poly L-lactide (PLLA) fibrous sheet (FS), which was coated with polydopamine for improved cell adhesion. The proliferation of hTMSCs within the hybrid-spheroids mixed with fragmented fibers was significantly increased as compared to that from the cell-only group. Cells and fragmented fibers were homogenously distributed with the presence of pore like empty spaces in the structure. LOX-1 staining revealed that the hybrid-spheroids improved the cell viability, which was potentially due to enhanced transport of oxygen through void space generated by engineered ECM. Transmission electron microscopy (TEM) analysis confirmed that cells within the hybrid-spheroid formed strong cell junctions and contacts with fragmented fibers. The expression of cell junction proteins including connexin 43 and E-cadherin was significantly upregulated in hybrid-spheroids by 16.53 ± 0.04 and 28.26 ± 0.11-fold greater than that from cell-only group. Similarly, expression of integrin α<SUB>2,</SUB> α<SUB>5</SUB>, and β<SUB>1</SUB> was significantly enhanced at the same group by 25.72 ± 0.13, 27.48 ± 0.49, and 592.78 ± 0.06-fold, respectively. In addition, stemness markers including Oct-4, Nanog, and Sox2 were significantly upregulated in hybrid-spheroids by 96.56 ± 0.06, 158.95 ± 0.06, and 115.46 ± 0.47-fold, respectively, relative to the cell-only group. Additionally, hTMSCs within the hybrid-spheroids showed significantly greater osteogenic differentiation under osteogenic media conditions. Taken together, our hybrid-spheroids can be an ideal approach for stem cell expansion and serve as a potential carrier for bone regeneration.</P> <P><B>Statement of Significance</B></P> <P>Cells are spatially arranged within extracellular matrix (ECM) and cell/ECM interactions are crucial for cellular functions. Here, we developed a hybrid-spheroid system incorporating engineered ECM prepared from fragmented electrospun fibers to tune stem cell functions. Conventionally prepared cell spheroids with large diameters (>200 µm) is often prone to hypoxia. In contrast, the hybrid-spheroids significantly enhanced viability and proliferation of human turbinate mesenchymal stem cells (hTMSCs) as compared to spheroid prepared from cell only. Under these conditions, the presence of fragmented fibers also improved maintenance of stemness of hTMSCs for longer time cultured in growth media and demonstrated significantly greater osteogenic differentiation under osteogenic media conditions. Thus, the hybrid-spheroids can be used as a delivery carrier for stem cell based therapy or a 3D culture model for <I>in vitro</I> assay.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>