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Biofabrication of organ-like engineered 3D tissue through the assembly of magnetized 3D multi-cellular spheroids has been recently investigated in tissue engineering. However, the cytotoxicity of magnetic nanoparticles (MNPs) and contraction-induced structural deformation of the constructs have been major limitations. In this study, we developed a method to fabricate composite stem cell spheroids using MNP-coated fibers, alleviating MNP-mediated toxicity and controlling structural assembly under external magnetic stimuli. The MNP-coated synthetic fibers (MSFs) were prepared by coating various amounts of MNPs on the fibers via electrostatic interactions. The MSFs showed magnetic hysteresis and no cytotoxicity on 2D-cultured adipose-derived stem cells (ADSCs). The composite spheroids containing MSFs and ADSCs were rapidly formed in which the amount of impregnated MSFs modulated the spheroid size. The fusion of in vitro composite spheroids was then monitored at the contacting interface; the fused spheroids with over 10 μg of MSF showed minimal contraction after 7 d, retaining around 90% of total area ratio regardless of the number of cells, indicating that the presence of fibers within the composite spheroid supported its structural maintenance. The fusion of MSF spheroids was modulated by external magnetic stimulation, and the effect of magnetic force on the movement and fusion of the spheroids was investigated using COMSOL simulation. Finally, ring and lamellar structures were successfully assembled using remote-controlled MSF spheroids, showing limited deformation and high viability up to 50 d during in vitro culture. In addition, the MSFs demonstrated no adverse effects on ADSC osteochondral differentiation. Altogether, we envision that our magnetic assembly system would be a promising method for the tissue engineering of structurally controlled organ-like constructs.

Biomaterials with graded functionality have various applications in cell and tissue engineering. In this study, by controlling oxidative polymerization of dopamine, we demonstrated universal techniques for generating chemical gradients on various materials with adaptability for secondary molecule immobilization. Diffusion-controlled oxygen supply was successfully exploited for coating of polydopamine (PD) in a gradient manner on different materials, regardless of their surface chemistry, which resulted in gradient in hydrophilicity and surface roughness. The PD gradient controlled graded adhesion and spreading of human mesenchymal stem cells (hMSCs) and endothelial cells. Furthermore, the PD gradient on these surfaces served as a template to allow for graded immobilization of different secondary biomolecules such as cell adhesive arginine-glycine-aspartate (RGD) peptides and siRNA lipidoid nanoparticles (sLNP) complex, for site-specific adhesion of human mesenchymal stem cells, and silencing of green fluorescent protein (GFP) expression on GFP-HeLa cells, respectively. In addition, the same approach was adapted for generation of nanofibers with surface in graded biomineralization under simulated body fluid (SBF). Collectively, oxygen-dependent generation of PD gradient on biomaterial substrates can serve as a simple and versatile platform that can be used for various applications realizing in vivo tissue regeneration and in vitro high-throughput screening of biomaterials.

Conductive biomaterials with a suitable biocompatibility have been utilized to fabricate in vitro platforms for differentiation of progenitor cell population as well as implantable tissue engineering scaffolds. This review evaluates biocompatibility of various conductive biomaterials and relevant fabrication techniques including coating, incorporation into composites, and functionalization with biological moieties. In addition, recent developments in tissue engineering applications using various conductive biomaterials are discussed in detail. Therefore, this overview could provide fundamental knowledge for engineering strategies in regulation of stem cell differentiation, maintenance of phenotypic characteristics, and design of functional implantable scaffolds for better regenerative medicines.

Although bone morphogenetic protein-2 (BMP-2) has been frequently used to stimulate bone formation, it has several side effects to be addressed, including the difficulty in optimization of clinically relevant doses and unwanted induction of cancerous signaling processes. In this study, an osteogenic peptide (OP) derived from BMP-2 was investigated as a substitute for BMP-2. In vitro studies showed that OP was able to enhance the osteogenic differentiation and mineralization of human mesenchymal stem cells (hMSCs). The peptides were then conjugated onto biocompatible poly-ι-lactide electrospun nanofibers through polydopamine chemistry. Surface chemical analysis proved that more than 80% of the peptides were stably retained on the nanofiber surface after 8 h of polydopamine coating during at least 28 days, and the amount of peptides that was retained increased depending on the polydopamine coating time. For instance, about 65% of the peptides were retained on nanofibers after 4 h of polydopamine coating. Also, a relatively small dose of peptides could effectively induce bone formation in in vivo critical-sized defects on the calvarial bones of mice. More than 50.4% ± 16.9% of newly formed bone was filled within the defect after treatment with only 10.5 ± 0.6 μg of peptides. Moreover, these groups had similar elastic moduli and contact hardnesses with host bone. Taken together, our results suggest that polydopamine-mediated OP immobilized on nanofibers can modulate the retention of relatively short lengths of peptides, which might make this an effective therapeutic remedy to guide bone regeneration using a relatively small amount of peptides.

Random skin flaps are commonly used in plastic and reconstructive surgery for patients suffering from severe or large scale wounds or in facial reconstruction. However, skin flaps are sometimes susceptible to partial or complete necrosis at the distal parts of the flaps due to insufficient blood perfusion in the defected area. In order to improve neovascularization in skin flaps, we developed an exogenous growth factor (GF) delivery platform comprised of coacervate-coated poly(lactic-co-glycolic acid) (PLGA) nanofibers. We used a coacervate that is a self-assembled complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., vascular endothelial growth factor (VEGF) and/or transforming growth factor beta 3 (TGF-β3)). The coacervate was coated onto a nanofibrous PLGA membrane for co-administration of dual GFs. In vitro proliferation of human dermal fibroblasts and endothelial tube formation using human umbilical vein endothelial cells indicated an enhanced bioactivity of released GFs when both VEGF and TGF-β3 were incorporated into coacervate-coated PLGA nanofibers (Coa-Dual NFs). Moreover, an in vivo study using a mouse skin flap model demonstrated that implantation of Coa-Dual NF reduced necrosis and enhanced blood perfusion in skin flap areas after 10 days, as compared to any single GF-loaded coacervate/PLGA fiber (Coa-Single NF) along with direct administration of the other GF onto the defect site. Moreover, Coa-Dual NFs exhibited a well-composed skin appendage and a significantly higher number of blood vessels. Based upon these results, we conclude that Coa-Dual NFs may stimulate cellular activity by enhancing the bioactivity of the released GF, leading to a synergetic effect of dual GFs for reducing necrosis in the random skin flaps. Therefore, Coa-Dual NFs could be a valuable drug delivery platform for a variety of potential clinical applications for skin tissue regeneration applications.

Bioengineered skin substitute offers new opportunities for treating various skin ailments. To compensate the structural integrity problems of scaffolds prepared from natural components, we mechanically developed highly modified electrospun nanofibrous membranes, incorporating poly(l-lactide-co-e-caprolactone) (PLCL) into gelatin [poly(l-lactide-co-e-caprolactone)/ gelatin membrane, (P/G (3/7)]. Subsequent to our previous in vitro study, our goal was to evaluate the in vivo performance of PLCL, gelatin, and P/G (3/7) membranes, and investigate the feasibility of the newly developed P/G (3/7) membrane for wound healing. Histological analysis using the mathematical model of wound healing and contraction, revealed the association between stiffness of skin substitute with cytokeratin production and wound contraction rate, and the defect site covered with the stiffer membrane showed lower cytokeratin production, and inversely, higher wound contraction rate. Overall, the P/G (3/7) membrane induced a satisfactory wound healing outcome. However, lower cytokeratin production rate with the mechanically modified P/G membrane involves the importance of the conditional blending of PLCL. Conversely, the condition of PLCL showed some incompatibility and hindrance of skin regeneration, consistent with previous in vitro results. With proper mechanical strength and cell viability, the P/G (3/7) membrane could successfully be used as a suitable skin substitute scaffold.

Polycaprolactone (PCL) has been considered a useful material for orthopedic devices and osseous implants because of its biocompatibility and bone-forming activity. However, PCL-based scaffolds have hydrophobic surfaces that reduce initial cell viability. In this study, we fabricated surface-modified PCL nanofibers for tissue engineering using radiation technology. We supplemented the hydrophilicity of the PCL nanofibers by introducing 2-aminoethyl methacrylate (AEMA) through gamma-irradiation and subsequently immobilized heparin onto the nanofibers using the EDC/NHS reaction. The SEM images show that there is almost no change in the morphology of nanofibers after radiation grafting of AEMA and heparin-immobilization onto PCL nanofibers. The surface properties of the scaffolds were characterized by ATR-FTIR, XPS, and fluorescamine staining in order to confirm the successful grafting of AEMA onto the PCL nanofibers. Immobilization of heparin was also confirmed by the amide I (1650 cm(-1)) and amide II group (1550 cm(-1)) from ATR-FTIR. The amounts of heparin were drastically increased on the AEMA-PCL nanofibers as revealed by TBO assay. The initial cell viability of hMSCs was significantly increased on the AEMA grafted nanofibers but grew slowly on heparin-immobilized nanofibers. The cumulative release of bone morphogenetic protein-2 (BMP-2) was slow and continuous onto the heparin-immobilized nanofibers (18.13 +/- 3.87 mu g mL(-1)) compared to PCL nanofibers (20.25 +/- 1.45 mu g mL(-1)). Therefore, heparin-immobilized nanofibers may be a good tool for tissue engineering applications using radiation technology.

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.

3D culture of stem cells can improve therapeutic effects. However, there is limited research on how to deliver cultured stem cell spheroids to the desired target. Here, we developed lotus seedpod-inspired hydrogel (LoSH) containing microwells for culture and delivery of stem cell spheroids. Human adipose-derived stem cells (hADSCs) inside the square microwells (200 or 400 μm in width with various depths) spontaneously formed spheroids with high viability (94.08 ± 1.56%), and fibronectins conjugated to the hydrogel successfully gripped the spheroids, similar to the funiculus gripping seeds in the lotus seedpod. The spheroids slightly bound to the LoSH surface at 37 °C were detached by the expansion of LoSH at lower temperature of 4 °C. After spheroid formation, LoSH was placed on the target substrate upside-down, expanded at 4 °C for 10 min, and removed from the target. As a result, the spheroids within the microwell were successfully transferred to the target substrate with high transfer efficiency (93.78 ± 2.30%). A delivery of spheroids from LoSH to full-thickness murine skin wound with chimney model showed significant enhancement of the number of SMA-positive vessels at day 21 compared to the group received the same number of spheroids by injection. Together, our findings demonstrate LoSH as a one-step platform that can culture and deliver spheroids to a large target area, which will be useful for various biomedical applications.

Myelination by oligodendrocytes (OLs) is a key developmental milestone in terms of the functions of the central nervous system (CNS). Demyelination caused by defects in OLs is a hallmark of several CNS disorders. Although a potential therapeutic strategy involves treatment with the myelin-forming cells, there is no readily available source of these cells. OLs can be differentiated from pluripotent stem cells; however, there is a lack of efficient culture systems that generate functional OLs. Here, we demonstrate biomimetic approaches to promote OL differentiation from human-induced pluripotent stem cells (iPSCs) and to enhance the maturation and myelination capabilities of iPSC-derived OL (iPSC-OL). Functionalization of culture substrates using the brain extracellular matrix (BEM) derived from decellularized human brain tissue enhanced the differentiation of iPSCs into myelin-expressing OLs. Co-culture of iPSC-OL with induced neuronal (iN) cells on BEM substrates, which closely mimics the in vivo brain microenvironment for myelinated neurons, not only enhanced myelination of iPSC-OL but also improved electrophysiological function of iN cells. BEM-functionalized aligned electrospun nanofibrous scaffolds further promoted the maturation of iPSC-OLs, enhanced the production of myelin sheath-like structures by the iPSC-OL, and enhanced the neurogenesis of iN cells. Thus, the biomimetic strategy presented here can generate functional OLs from stem cells and facilitate myelination by providing brain-specific biochemical, biophysical, and structural signals. Our system comprising stem cells and brain tissue from human sources could help in the establishment of human demyelination disease models and the development of regenerative cell therapy for myelin disorders.

EDITORIAL Tissue Engineering is proudly celebrating its 25th year in publication in 2019 and would like to begin this Editorial by personally thanking each and every one of our readers, contributors, reviewers, Editorial Board Members, and TERMIS leadership and membership for your continued support of the journal. Your impactful contributions, constructive reviews, and dedicated advocacy of the publication to your peers and institutional leadership has contributed to the continued success of our global publication and we look forward to celebrating our vast achievements throughout the year and beyond. Thank you for your continued support and advocacy.

Although many efforts have been made to engineer cell sheets with anisotropic patterns, current techniques still exhibit several shortcomings including uncontrolled harvest time and deformation of pattern by contraction. In this study, we report a simple method to harvest a cell sheet with a striated structure of extracellular matrix (ECM) and myoblasts using a thermosensitive hydrogel micropatterned with different sizes (25 and 80 μm). The hydrogel supported the formation of a confluent monolayer of myoblasts with aligned morphology. When the temperature was reduced from 37 to 4 oC, the size of the hydrogel increased by approximately 1.2-fold within 10 min. In response to this change, a cell sheet was harvested and could be transferred to the desirable substrate through conformal contact between the hydrogel and target. We further examined the effect of pattern size on alignment of ECM/cell assembly by fast Fourier transform (FFT) analysis of fluorescently stained stress fibers and ECM proteins within the harvested cell sheet. The cell sheet maintained alignment of confluent myoblasts after being harvested to the substrate. In addition, the topologic patterns also promoted formation of aligned myotube on the harvested cell sheet, which was important for muscle tissue regeneration. However, the pattern size appeared to have no influence on alignment of cells on the cell sheet. Finally, a bi-layered structure was fabricated in which each layer was stacked with aligned direction on the cell sheet being perpendicularly assembled. Collectively, our platform could be used for rapid harvest of cell sheets with aligned architecture of ECM/cell, which can be applied to fabrication of welldefined 3D tissue for tissue engineering.

Facile cell sheet translocation system is developed based on a thermally expandable hydrogel with modular cell adhesion favorable for both robust cell sheet formation and harvest. Efficient translocation is achieved at moderate cell-substrate interaction, which can be tuned by two-step reactions of mussel-inspired coating.

Skin flaps have the crucial issue that skin necrosis can occur due to tissue ischemia in the distal parts of flaps. To prevent tissue necrosis, skin flaps need to be supplied with sufficient blood perfusion through quick vascularization in the ischemic area. In this study, we fabricated poly(lactic-co-glycolic acid) (PLGA) meshes that had shaped random or alignments using electrospinning and modified the surface of the PLGA mesh, using 3,4-dihydroxy- L-phenylalanine (L-DOPA), for drug delivery. We carried out X-ray photoelectron spectroscopy and contact angle analysis to confirm the L-DOPA coating on the surface of PLGA meshes. Then, we loaded vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on the PLGA meshes for quicker angiogenesis through synergistic effect of the two growth factors. We investigated whether dual growth factor-loaded PLGA meshes on skin flaps promoted angiogenesis during 7-day treatment. When implanted into ICR mice models for skin flaps, the PLGA random mesh led to a result value of significantly smaller than the PLGA aligned mesh in skin flap necrosis and the epidermal thickness with the PLGA random mesh was much thicker than other groups in the necrotic area. Furthermore, coadministration of the two growth factors with the PLGA random meshes resulted in significantly higher blood vessel density than PLGA aligned mesh by ingrowth of host vessels at the edge of the skin flap. These results indicated that dual growth factor-loaded PLGA meshes could contribute to reducing the necrosis by dual growth factor delivery and geometric cues.

Multi-cellular spheroids are formed as a 3D structure with dense cell-cell/cell-extracellular matrix interactions, and thus, have been widely utilized as implantable therapeutics and various ex vivo tissue models in tissue engineering. In principle, spheroid culture methods maximize cell-cell cohesion and induce spontaneous cellular assembly while minimizing cellular interactions with substrates by using physical forces such as gravitational or centrifugal forces, protein-repellant biomaterials, and micro-structured surfaces. In addition, biofunctional materials including magnetic nanoparticles, polymer microspheres, and nanofiber particles are combined with cells to harvest composite spheroids, to accelerate spheroid formation, to increase the mechanical properties and viability of spheroids, and to direct differentiation of stem cells into desirable cell types. Biocompatible hydrogels are developed to produce microgels for the fabrication of size-controlled spheroids with high efficiency. Recently, spheroids have been further engineered to fabricate structurally and functionally reliable in vitro artificial 3D tissues of the desired shape with enhanced specific biological functions. This paper reviews the overall characteristics of spheroids and general/advanced spheroid culture techniques. Significant roles of functional biomaterials in advanced spheroid engineering with emphasis on the use of spheroids in the reconstruction of artificial 3D tissue for tissue engineering are also thoroughly discussed.

Although stem cell spheroids offer great potential as functional building blocks for bottom-up bone tissue engineering, delivery of bioactive signals remain challenging. Here, we engineered adenosine-ligand-modified fiber fragments to create a 3D cell-instructive microenvironment for bone. Briefly, the Poly(ι-lactic acid) (PLLA) nanofiber sheet was partially degraded into fragmented fibers (FFs) through aminolysis and adenosine was stably incorporated via one-step polydopamine coating. The SEM and XPS analysis demonstrated that polydopamine assisted adenosine coating efficiency was significantly increased, which led to high coating efficiency of adenosine and its significant retention. The engineered fibers were then assembled into stable spheroids with human-adipose-derived stem cells (hADSCs). The adenosine in the spheroids effectively stimulated A2bR (1.768 ± 0.08) signaling, which further significantly induced the expression of osteogenic markers such as Runx2 (3.216 ± 0.25), OPN (4.136 ± 0.14), OCN (10.16 ± 0.34), and OSX (2.27 ± 0.11) with improved mineral deposition (1.375 ± 0.05 μg per spheroid). In contrast, the adipogenic differentiation of hADSCs was significantly suppressed within the engineered spheroids. Transplantation of engineered spheroids strongly induced osteogenic differentiation of hADSCs in ectopic subcutaneous tissue. Finally, the bone regeneration was significantly enhanced by implanting AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). We propose that stem cell spheroids impregnated with engineered fibers enabling adenosine delivery could be promising building blocks for a bottom-up approach to create large tissues for regeneration of damaged bone.

Biomaterials with suitable surface modification strategies are contributing significantly to the rapid development of the field of bone tissue engineering. Despite these encouraging results, utilization of biomaterials is poorly translated to human clinical trials potentially due to lack of knowledge about the interaction between biomaterials and the body defense mechanism, the "immune system". The highly complex immune system involves the coordinated action of many immune cells that can produce various inflammatory and anti-inflammatory cytokines. Besides, bone fracture healing initiates with acute inflammation and may later transform to a regenerative or degenerative phase mainly due to the cross-talk between immune cells and other cells in the bone regeneration process. Among various immune cells, macrophages possess a significant role in the immune defense, where their polarization state plays a key role in the wound healing process. Growing evidence shows that the macrophage polarization state is highly sensitive to the biomaterial's physiochemical properties, and advances in biomaterial research now allow well controlled surface properties. This review provides an overview of biomaterial-mediated modulation of the immune response for regulating key bone regeneration events, such as osteogenesis, osteoclastogenesis, and inflammation, and it discusses how these strategies can be utilized for future bone tissue engineering applications.

In 2017, a new paradigm change caused by artificial intelligence and big data analysis resulted in innovation in each field of science and technology, and also significantly influenced progress in tissue engineering and regenerative medicine (TERM). TERM has continued to make technological advances based on interdisciplinary approaches and has contributed to the overall field of biomedical technology, including cancer biology, personalized medicine, development biology, and cell-based therapeutics. While researchers are aware that there is still a long way to go until TERM reaches the ultimate goal of patient treatment through clinical translation, the rapid progress in convergence studies led by technological improvements in TERM has been encouraging. In this review, we highlighted the significant advances made in TERM in 2017 (with an overlap of 5 months in 2016). We identified major progress in TERM in a manner similar to previous reviews published in the last few years. In addition, we carefully considered all four previous reviews during the selection process and chose main themes that minimize the duplication of the topics. Therefore, we have identified three areas that have been the focus of most journal publications in the TERM community in 2017: (i) advanced biomaterials and three-dimensional (3D) cell printing, (ii) exosomes as bioactive agents for regenerative medicine, and (iii) 3D culture in regenerative medicine.

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 NaHCO3 (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. In vitro 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 in vitro bone models, in vivo cell carriers, and as building blocks for bioprinting 3D bone tissue. Statement of Significance 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 in vitro and mineralized in the growth media. These spheroids were fused to form in vitro 3D mineralized tissue with larger size.

In this study, we developed a new system enabling rapid delivery of a multi-layered cell sheet by combining layer-by-layer (LBL) coating of a cell membrane and surface engineered thermally expandable hydrogel. Human dermal fibroblasts were LBL-coated with fibronectin (FN) and gelatin to form a multi-layered cell sheet in a single seeding step via spontaneous 3D cell-cell interactions. FN was covalently immobilized onto the surface of a Tetronic®-based hydrogel at two different concentrations (1 and 5 μg ml-1) for stable adhesion of the multi-layered cell sheet, followed by polydopamine coating. In both conditions, a multi-layered cell sheet was stably formed. Then, the cell sheet on the hydrogel modified with 1 μg ml-1 FN rapidly detached (>90% efficiency) in response to the expansion of the hydrogel when temperature changed from 37 °C to 4 °C, while the other group had a reduced detachment due to excessive cell-hydrogel interaction. The multi-layered cell sheet was evident in cell-extracellular matrix and cell-cell junction formation, and bFGF was continuously secreted over 7 days of in vitro culture. The multi-layered transplanted to the mouse subcutaneous tissue also exhibited evidence of vascular ingrowth, which collectively suggest that the delivery system maintaining cellular functions is applicable for regenerative medicine.

Regeneration of healthy endothelium onto vascular graft materials is imperative for prevention of intimal hyperplasia and thrombogenesis. In this study, we investigated the effect of collagen type IV (COL-IV) immobilized onto electrospun nanofibers on modulation of endothelial cell (EC) function, as a potential signal to rapid endothelialization of vascular grafts. COL-IV is assembled in basement membrane underneath intimal layer and regulates morphogenesis of blood vessels. For immobilization of COL-IV, poly(l-lactic acid) (PLLA) nanofibers (PL) were prepared as a model vascular graft substrate, onto which acrylic acid (AAc) was then grafted by using gamma-ray irradiation. AAc graft was dependent on irradiation doses and AAc concentrations, which allowed us to select the condition of 5% (v/v) AAc and 10 kGy for further conjugation of COL-IV. COL-IV immobilization was proportionally controlled as a function of its concentration. Atomic force microscope (AFM) analysis qualitatively supported immobilization of COL-IV, demonstrating increase in root mean square roughness of the PL from 665.37 ± 13.20 nm to 1440.74 ± 33.24. However, the Young's modulus of nanofibers was retained as approximately 1 MPa, regardless of surface modification. The number of ECs attached on the nanofibers with immobilized COL-IV was significantly increased by 5 times (1052 ± 138 cells/mm(2)) from pristine PL (234 ± 41 cells/mm(2)). In addition, the effect of immobilized COL-IV was profound for enhancing proliferation and up-regulation of markers implicated in rapid endothelialization. Collectively, our results suggest that COL-IV immobilized onto electrospun PLLA nanofibers may serve as a promising instructive cue used in vascular graft materials.

Engineering bone tissue is particularly challenging because of the distinctive structural features of bone within a complex biochemical environment. In the present study, we fabricated poly(L-lactic acid) (PLLA) electrospun nanofibers with random and aligned morphology immobilized with bone morphogenic protein-2 (BMP-2) and investigated how these signals modulate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and (2) in vivo bone growth rate, mechanical properties, and collagen assembly of newly formed bone. The orientation of adherent cells followed the underlying nanofiber morphology; however, nanofiber alignment did not show any difference in alkaline phosphate activity or in calcium mineralization of hMSCs after 14 days of in vitro culture in osteogenic differentiation media. In vivo bone regeneration was significantly higher in the nanofiber implanted groups (approximately 65-79%) as compared to the defect-only group (11.8 ± 0.2%), while no significant difference in bone regeneration was observed between random and aligned groups. However, nanoindentation studies of regenerated bone revealed Young's modulus and contact hardness with anisotropic feature for aligned group as compared to random group. More importantly, structural analysis of collagen at de novo bone showed the ability of nanofiber morphology to guide collagen deposition. SEM and TEM images revealed regular, highly ordered collagen assemblies on aligned nanofibers as compared to random fibers, which showed irregular, randomly organized collagen deposition. Taken together, we conclude that nanofibers in the presence of osteoinductive signals are a potent tool for bone regeneration, and nanofiber alignment can be used for engineering bone tissues with structurally assembled collagen fibers with defined direction.

In this work, we present a biomimetic fibrous scaffold containing two biomolecules. A biocompatible poly(L-lactide-co-e-caprolactone) mesh was fabricated by an electrospinning method, and then acrylic acid was grafted on the mesh to introduce a carboxyl group through γ-ray irradiation. Subsequently, the epidermal growth factor (EGF) and gelatin were coupled to the mesh through the EDC reaction. The modified mesh presents a consistent fibre diameter (874.4 ± 178.5 nm), with carboxyl groups (1.3 mM). EGF (171.7 ng mg−1 mesh) and gelatin (67.2 ± 30.5 μg mg−1 mesh) were successfully coupled on the mesh. The coupled EGF and gelatin promoted the cell viability 1.5-times higher than that from a non-modified mesh. In particular, the EGF on the meshes independently allowed hMSC to present a 3-times greater involucrin expression and enabled improved procollagen secretion, implying trans-differentiation of hMSC to keratinocyte-like cells. Therefore, the co-immobilization strategy of biomolecules using radiation technology may be an alternative tool for tissue engineering applications.

Stem cells are poorly permissive to non-viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell-binding ligand with a polymer that releases nucleic acids in a cytoplasm-responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9-derived hESC. Conjugating RVG to a redox-sensitive biodegradable dendrimer-type arginine-grafted polymer (PAM-ABP) enabled nanoparticle formation with plasmid DNA without altering the environment-sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG-PAM-ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ∼60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG-PAM-ABP is thus a novel bioreducible, biocompatible, non-toxic, synthetic gene delivery system for nAchR-expressing stem cells. Our data also demonstrates that a cell-binding ligand like RVG can cooperate with a gene delivery system like PAM-ABP to enable transfection of poorly-permissive cells.

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 α2, α5, and β1 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. Statement of Significance 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 in vitro assay.

Scaffold-free harvest of microtissue with a defined structure has received a great deal of interest in cell-based assay and regenerative medicine. In this study, we developed thermally expandable hydrogels with spatially controlled cell adhesive patterns for rapid harvest of geometrically controlled microtissue. We patterned polydopamine (PD) on to the hydrogel via microcontact printing (μCP), in linear shapes with widths of 50, 100 and 200 μm. The hydrogels facilitated formation of spatially controlled strip-like microtissue of human dermal fibroblasts (HDFBs). It was possible to harvest and translocate microtissues with controlled widths of 61.4 ± 14.7, 104.3 ± 15.6, and 186.6 ± 22.3 μm from the hydrogel to glass substrates by conformal contact upon expansion of the hydrogel in response to a temperature change from 37 to 4 °C, preserving high viability, extracellular matrix, and junction proteins. Microtissues were readily translocated in vivo to the subcutaneous tissue of mouse. The microtissues were further utilized as a simple assay model for monitoring of contraction in response to ROCK1 inhibitor. Collectively, micro-sized patterning of PD on the thermally expandable hydrogels via μCP holds promise for the development of microtissue harvesting systems that can be employed to ex vivo tissue assay and cell-based therapy. Statement of significance Harvest of artificial tissue with controlled cellular arrangement independently from external materials has been widely studied in cell-based assay and regenerative medicine. In this study, we developed scaffold-free harvest system of microtissues with anisotropic arrangement and controlled width by exploiting thermally expandable hydrogels with cell-adhesive patterns of polydopamine formed by simple microcontact printing. Cultured strips of human dermal fibroblasts on the hydrogels were rapidly delivered to various targets ranging from flat coverglass to mice subcutaneous tissue by thermal expansion of the hydrogel at 4 °C for 10 min. These were further utilized as a drug screening model responding to ROCK1 inhibitor, which imply its versatile applicability.

Although bone morphogenic proteins (BMPs) have been widely used for bone regeneration, the ideal delivery system with optimized dose and minimized side effects is still active area of research. In this study, we developed bone morphogenetic protein-2(BMP-2) immobilized poly(l-lactide) (PLLA) nanofibers inspired by polydopamine, which could be ultimately used as membranes for guided bone regeneration, and investigated their effect on guidance of in vitro cell behavior and in vivo bone formation. Surface chemical analysis of the nanofibers confirmed successful immobilization of BMP-2 mediated by polydopamine, and about 90% of BMP-2 was stably retained on the nanofiber surface for at least 28 days. The alkaline phosphatase activity and calcium mineralization of human mesenchymal stem cells (hMSCs) after 14 days of in vitro culture was significantly enhanced on nanofibers immobilized with BMP-2. More importantly, BMP-2 at a relatively small dose was highly active following implantation to the critical-sized defect in the cranium of mice; radiographic analysis demonstrated that 77.8 ± 11.7% of newly formed bone was filled within the defect for a BMP-2-immobilized groups at the concentration of 124 ± 9 ng/cm(2), as compared to 5.9 ± 1.0 and 34.1 ± 5.5% recovery, for a defect-only and a polydopamine-only group, respectively. Scanning and transmission electron microscopy of samples from the BMP-2 immobilized group showed fibroblasts and osteoblasts with nanofiber strands in the middle of regenerated bone tissue, revealing the importance of interaction between implanted nanofibers and the neighboring extracellular environment. Taken together, our data support that the presentation of BMP-2 on the surface of nanofibers as immobilized by utilizing polydopamine chemistry may be an effective method to direct bone growth at relatively low local concentration.

Human mesenchymal stem cells (hMSCs) have the ability to differentiate into mesenchymal lineages. In this study, we hypothesized that treatment of embryoid bodies (EBs) composed of either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) with a hMSC-conditioned medium (CM) can stimulate the induction of the mesodermal lineage and subsequent differentiation toward the osteogenic and chondrogenic lineage. Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) analysis indicated that the hMSC-CM treatment increased gene expression related to the mesodermal lineage and decreased gene expression related to the endodermal and ectodermal lineage in EBs. Fourteen days after culturing the mesodermal lineage-induced EBs in the osteogenic or chondrogenic differentiation medium, we observed enhanced osteogenic and chondrogenic differentiation compared with untreated EBs, as evaluated using qRT-PCR, cytochemistry, immunocytochemistry, and flow cytometry. This method may be useful for enhancing the osteogenic or chondrogenic differentiation of hESCs or hiPSCs.

Recently, stem cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stem cell spheroids in different sizes from micro-scaled cell sheets (μCS) using thermosensitive hydrogels and investigated their effects on stem cell function. Mesenchymal stem cells isolated from different tissues such as human turbinate tissue, bone marrow, and adipose tissue were adhered selectively to each micro-pattern (squares with widths of 100 and 400 μm) on the surface of the hydrogel and formed μCS. The diameters of the spheroids were modulated by the size of the patterns (45±5 and 129±4 μm in diameter for the 100 and 400 μm micro-patterns, respectively) and the seeding density (129±4, 149±6, and 163±6 μm for 5.0, 10.0, and 15.0 × 10alt;supagt;4alt;/supagt; cells/cmalt;supagt;2alt;/supagt;, respectively, on 400-μm micro-pattern). In addition, the spheroids were successfully fabricated regardless of stem cell origin, and the diameter of the spheroids was also affected by cell spreading area on a cell culture dish. Stemness markers were highly expressed in the spheroids regardless of the spheroid size. Furthermore, an increase in E-cadherin and decrease in N-cadherin gene expression showed the stable formation of spheroids of different sizes. Gene expression levels of hypoxia inducible factors and secretion of vascular endothelial growth factor (VEGF) were increased (13.2±1.4, 325±83.4 and 534.3±121.5 pg/ng DNA in a monolayer, and 100- and 400-μm micro-patterned spheroids, respectively) proportional to the diameters of the spheroids. The size of spheroids were maintained even after injection, cryopreservation and 7 days of suspension culture with high viability (~90%). In conclusion, this novel technique to fabricate spheroids with controlled size could be widely applied in various applications that require a controlled size in regenerative medicine.

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L-lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose-derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG-coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52 ± 28.10%) as compared to defect (17.48 ± 11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.

Novel freeze-dried porous composite scaffolds were prepared from natural polymer of silk fibroin (SF) as a matrix and from diopside nanoceramic as a bioactive reinforcing agent through a freeze-drying method. Considering the superiority of both diopside and SF in terms of mechanical and biochemical properties (in comparison to similar ceramics and polymers, especially in maxillofacial specific applications), we investigated the effect of diopside nanoparticle contents. Microstructure studies illustrated that the bioactive diopside nanoparticles were distributed throughout the fibroin matrix. Enough porosity, desired surface properties, high mechanical strength and excellent in vitro biocompatibility were achieved during the fabrication of the nanocomposite scaffolds without any extra modifications.

The concept of guided bone regeneration facilitated by barrier membranes has been widely considered to achieve enhanced bone healing in maxillofacial surgery. However, the currently available membranes are limited in their active regulation of cellular activities. In this study, we fabricated polycaprolactone/gelatin composite electrospun nanofibers incorporated with basic fibroblast growth factor (bFGF) to direct bone regeneration. The fibrous morphology was maintained after the crosslinking and subsequent conjugation of heparin. Release of bFGF from electrospun nanofibers without heparin resulted in a spontaneous burst, while the heparin-mediated release of bFGF decreased the burst release in 24 h. The bFGF released from the nanofibers enhanced the proliferation and migration of human mesenchymal stem cells as well as the tubule formation of human umbilical cord blood cells. The subcutaneous implantation of fibers incorporated with bFGF mobilized a large number of cells positive for CD31 and smooth muscle alpha actin within 2 weeks. The effect of the nanofibers incorporated with bFGF on bone regeneration was evaluated on a calvarial critical size defect model. As compared to the mice that received fibers without bFGF, which presented minimal new bone formation (5.36 ± 3.4 % of the defect), those that received implants of heparinized nanofibers incorporated with 50 or 100 ng/mL bFGF significantly enhanced new bone formation (10.82 ± 2.2 and 17.55 ± 6.08 %). Taken together, our results suggest that the electrospun nanofibers incorporating bFGF have the potential to be used as an advanced membrane that actively enhances bone regeneration.

Recently, it was reported that bone morphogenetic protein 4 (BMP4) alone or BMP4 combined with fibroblast growth factor 2 (FGF2) treatment enhanced mesodermal differentiation of human embryonic stem cells (hESCs) that were cultured feeder-free on Matrigel. In this study, we show that mesodermal lineage-induced embryoid bodies (EBs) generate greater numbers of osteogenic and chondrogenic lineage cells. To induce the mesodermal lineage, hESCs were treated with BMP4 and FGF2 during the EB state. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that the treatment decreased endodermal and ectodermal lineage gene expression and increased mesodermal lineage gene expression. Importantly, the mesodermal lineage-induced EBs underwent enhanced osteogenic and chondrogenic differentiation after differentiation induction. This method could be useful to enhance the osteogenic or chondrogenic differentiation of hESCs.

Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5 × 105 cells/mm2 was used, the thickness of assembly was 41.90 ± 1.69 μm, with approximately 9.3 ± 1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution-mediated transfection are limited due to low transfection efficiency and insufficient duration of cell-siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio-inspired polymer-mediated reverse transfection system is developed consisting of implantable poly(lactic-co-glycolic acid) (PLGA) scaffolds functionalized with siRNA-lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA-coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA-sLNP-PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose-derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA-sLNP-PLGA scaffolds enhanced bone formation in a mouse model of critical-sized bone defect. Therefore, the bio-inspired reverse transfection system can provide an all-in-one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.

Developing an artificial extracellular matrix that closely mimics the native tissue microenvironment is important for use as both a cell culture platform for controlling cell fate and an in vitro model system for investigating the role of the cellular microenvironment. Electrospinning, one of the methods for fabricating structures that mimic the native ECM, is a promising technique for creating fibrous platforms. It is well-known that align or randomly distributed electrospun fibers provide cellular contact guidance in a single pattern. However, native tissues have hierarchical structures, i.e., topographies on the micro- and nanoscales, rather than a single structure. Thus, we fabricated randomly distributed nanofibrous (720 ± 80 nm in diameter) platforms via a conventional electrospinning process, and then we generated microscale grooves using a femtosecond laser ablation process to develop engineered fibrous platforms with patterned hierarchical topographies. The engineered fibrous platforms can regulate cellular adhesive morphology, proliferation, and distinct distribution of focal adhesion proteins. Furthermore, confluent myoblasts cultured on the engineered fibrous platforms revealed that the direction of myotube assembly can be controlled. These results indicate that our engineered fibrous platforms may be useful tools in investigating the roles of nano- and microscale topographies in the communication between cells and ECM.

Surface modification of tissue engineering scaffolds and substrates is required for improving the efficacy of stem cell therapy by generating physicochemical stimulation promoting proliferation and differentiation of stem cells. However, typical surface modification methods including chemical conjugation or physical absorption have several limitations such as multistep, complicated procedures, surface denaturation, batch-to-batch inconsistencies, and low surface conjugation efficiency. In this study, we report a mussel-inspired, biomimetic approach to surface modification for efficient and reliable manipulation of human neural stem cell (NSC) differentiation and proliferation. Our study demonstrates that polydopamine coating facilitates highly efficient, simple immobilization of neurotrophic growth factors and adhesion peptides onto polymer substrates. The growth factor or peptide-immobilized substrates greatly enhance differentiation and proliferation of human NSCs (human fetal brain-derived NSCs and human induced pluripotent stem cell-derived NSCs) at a level comparable or greater than currently available animal-derived coating materials (Matrigel) with safety issues. Therefore, polydopamine-mediated surface modification can provide a versatile platform technology for developing chemically defined, safe, functional substrates and scaffolds for therapeutic applications of human NSCs.

Synthetic substrates with defined chemical and structural characteristics may potentially be prepared to mimic the living ECM to regulate cell adhesion and growth. Hydrogels with cell-adhesive peptides (0.28 ± 0.03 nmol peptide cm(-2) , TTA-R-0.5; and 0.91 ± 0.12 nmol peptide cm(-2) , TTA-R-2.0) and/or micro-scaled topographical patterns (10, 25, and 80 µm grooves) are prepared using enzymatic polymerization. The adherent morphology and proliferation of C2C12 skeletal myoblasts and human aortic smooth muscle cells (hAoSM) on the hydrogels are studied. The newly developed hydrogels may be useful in investigating the roles of cell adhesion and substrate surface properties in the communication of adherent cells with the ECM.

The structure of tissue plays a critical role in its function and therefore a great deal of attention has been focused on engineering native tissue-like constructs for tissue engineering applications. Transfer printing of cell layers is a new technology that allows controlled transfer of cell layers cultured on smart substrates with defined shape and size onto tissue-specific defect sites. Here, the temperature-responsive swelling-deswelling of the hydrogels with groove patterns and their versatile and simple use as a template to harvest cell layers with anisotropic extracellular matrix assembly is reported. The hydrogels with a cell-interactive peptide and anisotropic groove patterns are obtained via enzymatic polymerization. The results show that the cell layer with patterns can be easily transferred to new substrates by lowering the temperature. In addition, multiple cell layers are stacked on the new substrate in a hierarchical manner and the cell layer is easily transplanted onto a subcutaneous region. These results indicate that the evaluated hydrogel can be used as a novel substrate for transfer printing of artificial tissue constructs with controlled structural integrity, which may hold potential to engineer tissue that can closely mimic native tissue architecture.

Sulphur-containing spring water, which is generally rich in minerals and metallic elements, is tradition- ally considered beneficial for diseases of dermal origin. We investigated sulphur-containing spring water from the Ildong Jeil springs in Korea for antibacterial properties as well as antioxidant and cytoprotective effects on primary human dermal fibroblasts. Sulphurous spring water from Ildong Jeil springs significantly restricted bacterial growth compared to that of spring water with no sulphur content. We also recorded significant increases in total cellular anti-oxidant activity in primary hu- man dermal fibroblasts cultured in media reconstituted in sulphurous spring water. Although, procollagen gene expression was not enhanced significantly, culture medium prepared using sulphurous spring water protected dermal fibroblasts from tBOOH (tertiary butyl hydroperoxide)-induced cytotoxicity. Our data provides evidence that sulphur-containing spring water has beneficial effects on primary human dermal fibroblasts substantiating its medicinal properties on human skin.

The uniform nanofibers of poly(L-lactide---caprolactone) (PLCL) with different contents of marine collagen (MC) were successfully prepared by electrospinning method. The effects of the major parameters in electrospinning process such as tip to target distance (TTD), voltage, nozzle size and flow rate on the average diameter of the electrospun nanofiber were investigated in generating composite nanofiber. The diameter and morphology of the nanofibers were confirmed by a scanning electron microscopy (SEM). Also, we measured a water contact angle to determine the surface wettability of the nanofibers. The average diameter of the nanofibers decreased as the value of TTD, MC contents, and voltages increased in comparison with that of pristine PLCL nanofiber. In contrast, the diameter of the nanofibers increased as the flow rate and inner diameter of nozzle increased in comparison with that of pristine PLCL. In addition, the hydrophilicity of the nanofiber and attachment of MG-63 cells on the sheets increased as incorporated collagen contents increased. Therefore, the marine collagen would be a potential material to enhance cellular interactivity of synthetic materials by mimicking the natural tissue.

Biomimetic scaffolds mimic important features of the extracellular matrix (ECM) architecture and can be finely controlled at the nano- or microscale for tissue engineering. Rational design of biomimetic scaffolds is based on consideration of the ECM as a natural scaffold; the ECM provides cells with a variety of physical, chemical, and biological cues that affect cell growth and function. There are a number of approaches available to create 3D biomimetic scaffolds with control over their physical and mechanical properties, cell adhesion, and the temporal and spatial release of growth factors. Here, an overview of some biological features of the natural ECM is presented and a variety of original engineering methods that are currently used to produce synthetic polymer-based scaffolds in pre-fabricated form before implantation, to modify their surfaces with biochemical ligands, to incorporate growth factors, and to control their nano- and microscale geometry to create biomimetic scaffolds are discussed. Finally, in contrast to pre-fabricated scaffolds composed of synthetic polymers, injectable biomimetic scaffolds based on either genetically engineered- or chemically synthesized-peptides of which sequences are derived from the natural ECM are discussed. The presence of defined peptide sequences can trigger in situ hydrogelation via molecular self-assembly and chemical crosslinking. A basic understanding of the entire spectrum of biomimetic scaffolds provides insight into how they can potentially be used in diverse tissue engineering, regenerative medicine, and drug delivery applications.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.

Blends of PAni and PLCL are electrospun to prepare uniform fibers for the development of electrically conductive, engineered nerve grafts. PC12 cell viability is significantly higher on RPACL fibers than on PLCL-only fibers, and the electrical conductivity of the fibers affects the differentiation of PC12 cells; the number of cells positively-stained and their expression level are significantly higher on RPACL fibers. PC12 cell bodies display an oriented morphology with outgrowing neurites. On RPACL fibers, the expression level of paxillin, cdc-42, and rac is positively affected and proteins including RhoA and ERK exist as more activated state. These results suggest that electroactive fibers may hold promise as a guidance scaffold for neuronal tissue engineering.

The development of an artificial matrix is critical as both a substrate to control the cell behavior and as a tool for examining the roles of cellular microenvironment in biology. This study developed cell-interactive hydrogels containing a Arg-Gly-Asp (RGD) peptide, cross-linked via bio-inspired enzymatic processes using H2O2 and horseradish peroxidase (HRP) as the initiators and examined how they controlled myoblast functions. The cell-interactive hydrogels modulated the adhesion and proliferation of myoblast, depending on the peptide density. Furthermore, the expression of focal adhesion proteins and myogenic differentiation were up-regulated significantly in myoblasts cultured on peptide-incorporated hydrogels. Therefore, the novel hydrogel system can be used to regulate the cell function for many tissue engineering applications.

A variety of surface modification techniques have been proposed to improve the cell-biomaterial interactions. On the other hand, these processes may cleave long-chained polymers, and compromise their mechanical properties. In this study, dopamine was used as a bridge molecule to immobilize gelatin on the poly(L-lactide-co-ɛ-caprolactone) (PLCL) fibrous matrices, which may then be used as a cell delivery carrier. The PLCL fibrous matrices coated with polydopamine by dipping (D-PLCL) can subsequently immobilize gelatin (GD-PLCL). The D-PLCL matrices showed minimal changes in the mechanical properties with a tensile strain of 251.0±33.4% and 247.8±32.1% before and after the coating process, respectively. The cellular activities on the fibrous matrices increased in the order of PLCL