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Background Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation. Methods We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s t-test (for two variables) (P Results Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063 mm2–0.322 mm2). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA). Conclusion The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications.

In tissue engineering, excessively generated reactive oxygen species (ROS) during biomaterial implantation or cell transplantation is a one of major causes of diminishing therapeutic effects. In this study, we prepared biomaterial surfaces coated with antioxidant epigallocatechin gallate (EGCG) and metal ions, and evaluated their anti-oxidative and ROS scavenging properties. We revealed that EGCG-coating on polycaprolactone (PCL) film surface increased hydrophilicity and anti-oxidative properties as a function of total phenol content (TPC) potentially due to the increase in phenolic -OH and π-electrons from structural maintenance and directly removed the hydrogen peroxide (H2O2) by resonance-stabilization. Furthermore, EGCG-coated PCL film increased attachment, spreading area, and viability of human adipose-derived stem cells (hADSCs) against H2O2 treatment while stimulated the cellular signaling to reduce apoptotic gene and enhance anti-oxidative enzyme expression. Further, we applied EGCG coating on the surface of poly-L-lactic acid (PLLA) fibers. Spheroids incorporating EGCG-coated PLLA fibers were able to maintain their shape and showed improved viability and anti-oxidative activities in response to H2O2-induced oxidative stress than control spheroids. Therefore, metal-phenolic network (MPN) coating of EGCG is a suitable method to impart the anti-oxidative properties to biomaterials by evaluating the structural properties and biological effects.

Guided bone regeneration refers to the process in which bone defects could be regenerated by facilitated healing through the use of membranes, potentially with the delivery of osteoinductive molecules, however, the regeneration often failed due to inflammation during bone formation. In this study, we developed a membrane immobilized with lactoferrin to modulate both bone regeneration and inflammatory responses. Lactoferrin was immobilized on electrospun nanofibers (LF50) by exploiting an adhesive polydopamine coating method. When human adipose-derived stem cells (hADSCs) were seeded onto the nanofibers, the LF50 significantly increased the osteogenic differentiation. For example, the gene expression of osteopontin was 6.9 ± 2.3 times greater in the cells on LF50 than the cells on unmodified nanofibers without lactoferrin. In addition, the gene expression of tumor necrosis factor-alpha (

The goal of this study was to investigate the effect of transplantation of an adipose-derived stem cell (ASC) spheroid sheet on the regeneration of ulcerated oral mucosa. The ASC spheroid sheet was constructed by seeding three-dimensional (3D) spheroidal ASCs on a temperature-responsive hydrogel, where cell adhesion and spreading were improved by incorporating fibronectin. The 3D culture of ASCs enhanced their inherent paracrine properties for up to 1 week after transferring to two-dimensional (2D) culture conditions. Expansion of the hydrogel through temperature change promoted effective delivery of ASCs to the site of injury, and viability was maintained after transferring ASCs to the target substrate. Treatment with the ASC spheroid sheet accelerated wound closure and regeneration of the stratified multi-layered epithelium. The regenerated epithelium not only had structural integrity, but also displayed phenotypic polarization across the mucosa; basal epithelial cells expressed cytokeratin 5, whereas cytokeratin 13-positive cells were located in the supra-basal layers. In addition, by day 7 after treatment, the mucosa that received the ASC spheroid sheet had significantly decreased inflammation, similar to that of the normal mucosa. These results demonstrate that the ASC spheroid sheet is an effective treatment for ulcers in the oral cavity.

Scaffold porosity has played a key role in bone tissue engineering aimed at effective tissue regeneration, by promoting cell attachment, proliferation, and osteogenic differentiation for new bone formation. Three-dimensional plotting systems (3DPSs) have been widely used to introduce porosity to the scaffold; however, introducing certain features in the scaffold strands that improve bone tissue regeneration remains a challenge. In this work, we fabricated bone tissue scaffolds with macro- and microporous structural features using a 3DPS and non-solvent-induced phase separation method. This approach allowed both macro- and micropores to be created in the scaffold strands. The surface morphology and mechanical and degradation properties of the perforated scaffolds were characterized carefully. Human marrow stromal cells were cultured on the scaffolds and then analyzed in vitro to assess scaffold bio-function. The highly porous scaffold exhibited mechanical properties similar to those of cancellous bone. Cell attachment, proliferation, and differentiation were significantly higher in porous scaffold compared to its nonporous counterpart. These results suggest that highly porous scaffolds have tremendous potential as a bone tissue regeneration platform.

The use of engineered scaffolds or stem cells is investigated widely in the repair of injured musculoskeletal tissue. However, the combined regeneration of hierarchical osteochondral tissue remains a challenge due to delamination between cartilage and subchondral bone or difficulty in spatial control over differentiation of transplanted stem cells. Here, two types of composite spheroids are prepared using adipose‐derived stem cells (hADSCs) and nanofibers coated with either transforming growth factor‐β3 or bone morphogenetic growth factor‐2 for chondrogenesis or osteogenesis, respectively. Each type of spheroid is then cultured within a 3D‐printed microchamber in a spatially arranged manner to recapitulate the bilayer structure of osteochondral tissue. The presence of inductive factors regionally modulates in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct without dedifferentiation. Furthermore, hADSCs from each spheroid proliferate and sprout and successfully connect the two layers mimicking the osteochondral interface without apertures. In vivo transplantation of the biphasic construct onto a femoral trochlear groove defect in rabbit knee joint results in 21.2 ± 2.8% subchondral bone volume/total volume and a cartilage score of 25.0 ± 3.7. The present approach can be an effective therapeutic platform to engineer complex tissue., The spontaneously differentiated chondrogenic or osteogenic composite spheroids incorporating growth factors‐engineered fibers are spatially positioned onto each cell‐adhesive 3D printed microchamber, and the chambers are then hierarchically assembled for the biphasic construct to mimic an osteochondral tissue structure. The biphasic construct is then transplanted onto an osteochondral defect for in vivo analysis.

Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (1 kPa), has not yet been studied. In this study, we determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction.

In this study, we prepared polydopamine-gelatin templated electrospun nanofibers, which were coated with precisely controlled amounts of biominerals in the form of simulated body fluid (SBF). We initially investigated the effects of different mineral concentrations on the in vitro osteogenic differentiation of human adipose derived stem cells (hADSCs), demonstrating the increase in osteogenic differentiation as a function of mineral concentration without any osteogenic supplements. Meanwhile, in the presence of chondrogenic medium, nanofibers with lower mineral contents showed the highest chondrogenic commitment of hADSCs, while more highly mineralized samples greatly inhibited chondrogenesis. We then generated a mineral gradient on the nanofiber surface and examined the hADSC responses under different media conditions. The hADSCs showed a gradient osteoblast phenotype on a mineral gradient of nanofibers under growth media conditions. Under chondrogenic media conditions, hADSCs favored osteogenic differentiation in the more mineralized positions, while lower mineral content induced chondrogenic differentiation of the hADSCs, consistent with the trend in nanofibers with different mineral contents. These results suggest that the controlled presentation of minerals on the material substrate in combination with soluble inducing factors could be used as a tool for stem cell-based osteochondral tissue engineering.

Cell spheroids as building blocks for engineering micro-tissue should be able to mimic the complex structure of natural tissue. However, control of the distribution of multiple cell populations within cell spheroids is difficult to achieve with current spheroid-harvest methods such as hanging-drop and with the use of microwell plates. In this study, we report the fabrication of core-shell spheroids with the ultimate goal to form 3D complex micro-tissue. We used endothelial cells and two types of stem cells (human turbinate mesenchymal stem cells (hTMSCs)/adipose-derived stem cells (ADSCs)). The stem cells and endothelial cells formed layered micro-sized cell sheets (µCSs) on polydopamine micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids by expansion of the hydrogels. The co-cultured spheroids formed a core-shell structure irrespective of stem cell type. In addition, the size of the core-shell spheroids was controlled from 90 ± 1 to 144 ± 3 µm by changing pattern sizes (200, 300, and 400 µm). The shell thickness gradually increased from 12 ± 3 to 30 ± 6 µm by adjusting the endothelial cell seeding density. Finally, we fabricated the micro-tissue by fusion of the co-cultured spheroids, and the spheroids with the core-shell structure rapidly induced in vitro vessel-like network in 3 days. Thus, the position of endothelial cells in co-cultured spheroids may be an important factor for the modulation of the vascularization process, which can be useful for the production of 3D complex micro-tissues using spheroids as building blocks. Statement of significance This manuscript describes our work on the fabrication of core-shell spheroids as building blocks to form 3D complex vascularized micro-tissue. Stem cells (human turbinate mesenchymal stem cells (hTMSCs) or adipose-derived stem cells (ADSCs)) and endothelial cells formed layered micro-sized cell sheets (µCSs) on micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids (core – stem cells, shell – endothelial cells), irrespective of stem cell type. In addition, the size and shell thickness of the core-shell spheroids were controlled by modifying pattern size and endothelial cell seeding density. We fabricated the vascularized micro-tissue by fusion of the spheroids and demonstrated that the spheroids with a core-shell structure rapidly induced vessel-like network.

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

Stem cell spheroids have been widely investigated to accelerate bone tissue regeneration. However, the directed differentiation of stem cells into osteoblastic lineage and the prevention of cells from damage by reactive oxygen species (ROS) remain challenge. Here, we developed osteoinductive and ROS scavenging extracellular matrix-mimicking synthetic fibers based on epigallocatechin gallate (EGCG) coating. They were then utilized to fabricate engineered spheroids with human adipose-derived stem cells (hADSCs) for bone tissue regeneration. The EGCG-mineral fibers (EMF) effectively conferred osteoinductive and ROS scavenging signals on the hADSCs within spheroids, demonstrating relative upregulation of antioxidant genes (SOD-1 (25.8 ± 2.1) and GPX-1 (3.3 ± 0.1) and greater level of expression of osteogenic markers, runt-related transcription factor (5.8 ± 0.1) and osteopontin (5.9 ± 0.1), compared to hADSCs in the spheroids without EMF. The in vitro overexpression of osteogenic genes from hADSCs was achieved from absence of osteogenic supplements. Furthermore, in vivo transplantation of hADSCs spheroids with the EMF significantly promoted calvarial bone regeneration (48.39 ± 9.24%) compared to that from defect only (17.38 ± 6.63%), suggesting that the stem cell spheroid biofabrication system with our novel mineralization method described here is a promising tool for bone tissue regeneration.

Three-dimensional (3D)-printed scaffolds have proved to be effective tools for delivering growth factors and cells in bone-tissue engineering. However, delivering spheroids that enhance cellular function remains challenging because the spheroids tend to suffer from low viability, which limits bone regeneration in vivo. Here, we describe a 3D-printed polycaprolactone micro-chamber that can deliver human adipose-derived stem cell spheroids. An in vitro culture of cells from spheroids in the micro-chamber exhibited greater viability and proliferation compared with cells cultured without the chamber. We coated the surface of the chamber with 500 ng of platelet-derived growth factors (PDGFs), and immobilized 50 ng of bone morphogenetic protein 2 (BMP-2) on fragmented fibers, which were incorporated within the spheroids as a new platform for a dual-growth-factor delivery system. The PDGF detached from the chamber within 8 h and the remains were retained on the surface of chamber while the BMP-2 was entrapped by the spheroid. In vitro osteogenic differentiation of the cells from the spheroids in the micro-chamber with dual growth factors enhanced alkaline phosphatase and collagen type 1A expression by factors of 126.7 ± 19.6 and 89.7 ± 0.3, respectively, compared with expression in a micro-chamber with no growth factors. In vivo transplantation of the chambers with dual growth factors into mouse calvarial defects resulted in a 77.0 ± 15.9% of regenerated bone area, while the chamber without growth factors and a defect-only group achieved 7.6 ± 3.9% and 5.0 ± 1.9% of regenerated bone areas, respectively. These findings indicate that a spheroid-loaded micro-chamber supplied with dual growth factors can serve as an effective protein-delivery platform that increases stem-cell functioning and bone regeneration.

Adenosine and its receptors have emerged as alternative targets to control cellular functions for bone healing. However, the soluble delivery of adenosine has not proven effective because of its fast degradation in vivo. We therefore designed a stable coating of adenosine for biomaterial surfaces through polydopamine chemistry to control osteogenesis and osteoclastogenesis via A2bR signaling. First, we prepared electrospun poly (ι-lactic acid) (PLLA) nanofiber sheets, which were modified through a one-step adenosine polydopamine coating process. Scanning electron microscopy (SEM) revealed deposition of particles on the adenosine polydopamine-coated PLLA (AP-PL) sheets compared to the polydopamine-only sheets. Moreover, X-ray photoelectron spectroscopy analysis confirmed an increase in nitrogen signals due to adenosine. Furthermore, adenosine loading efficiency and retention were significantly enhanced in AP-PL sheets compared to polydopamine-only sheets. Human adipose-derived stem cells (hADSCs) cultured on AP-PL expressed A2bR (1.30 ± 0.19 fold) at significantly higher levels than those cultured on polydopamine-only sheets. This in turn significantly elevated the expression of Runx2 (16.94 ± 1.68 and 51.69 ± 0.07 fold), OPN (1.63 ± 0.16 and 30.56 ± 0.25 fold), OCN (1.16 ± 0.13 and 5.23 ± 0.16 fold), and OSX (10.01 ± 0.81 and 62.48 ± 0.25 fold) in cells grown in growth media on days 14 and 21, respectively. Similarly, mineral deposition was enhanced to a greater extent in the AP-PL group than the polydopamine group, while blocking of A2bR significantly downregulated osteogenesis. Finally, osteoclast differentiation of RAW 264.7 cells was significantly inhibited by growth on AP-PL sheets. However, osteoclast differentiation was significantly stimulated after A2bR was blocked. Taken together, we propose that polydopamine-assisted one-step coating of adenosine is a viable method for surface modification of biomaterials to control osteogenic differentiation of stem cells and bone healing.

Stimuli-responsive materials have been a major subject of investigation for cell sheet technology in regenerative medicine and pharmaceutical research. Most materials and processes, however, have shortcomings, such as a relatively long operation time, varied detachment efficiency, and potential toxicity. In this study, we develop an effective cell sheet translocation protocol using a cell adhesive and temperature-responsive hydrogel. Cell adhesion on the hydrogel was modulated with a bio-inspired coating of polydopamine (PD), the amount of which was tuned to increase as a function of coating time (30–120 min). This PD-coated hydrogel promotes the adhesion of human dermal fibroblasts (HDFBs), mediated by serum protein adsorption, and allows for the formation of a cell sheet (confluent cell monolayer) following culture. Hydrogel cell sheets with 30 min of PD coating (PD30) were readily translocated to a target surface (glass) within 10 min of thermal expansion (induced by changing the temperature from 37 to 4 °C). Under these conditions, the translocation efficiency and cell viability were greater than 90%. However, hydrogel cell sheets with >60 min PD coating remained almost completely attached, while the surfaces exhibited significantly lower cell viability (

Although bone grafting with autologous or allogeneic sources still remains as a gold standard, there is high demand for alternative approach for the treatment of patients. Various biomaterials including natural or synthetic materials, and their composite have been utilized for rapid bone regeneration. However, conventional biomaterials are not ideal and those enabling simultaneously provision of structural support and active induction of tissue regeneration are needed. In particular, frontiers in bone biomaterials have been designed with capability of delivering biologic, structural, and compositional features of natural bone extracellular matrix microenvironment. In this review, we categorize and summarize representative approaches.

In tissue engineering, excessively generated reactive oxygen species (ROS) during biomaterial implantation or cell transplantation is a one of major causes of diminishing therapeutic effects. In this study, we prepared biomaterial surfaces coated with antioxidant epigallocatechin gallate (EGCG) and metal ions, and evaluated their anti-oxidative and ROS scavenging properties. We revealed that EGCG-coating on polycaprolactone (PCL) film surface increased hydrophilicity and anti-oxidative properties as a function of the coated amount of EGCG potentially due to the increase in phenolic -OH and π-electrons from structural maintenance and directly removed the hydrogen peroxide (H2O2) by resonance-stabilization. Furthermore, EGCG-coated PCL film increased attachment, spreading area, and viability of human adipose-derived stem cells (hADSCs) against H2O2 treatment while stimulated the cellular signaling to reduce apoptotic gene and enhance anti-oxidative enzyme expression. Further, we applied EGCG coating on the surface of poly-L-lactic acid (PLLA) fibers. Spheroids incorporating EGCG-coated PLLA fibers were able to maintain their shape and showed improved viability and anti-oxidative activities in response to H2O2-induced oxidative stress than control spheroids. Therefore, MPN coating of EGCG is a suitable method to impart the anti-oxidative properties to biomaterials by evaluating the structural properties and biological effects.

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.

Numerous methods have been reported for the fabrication of 3D multi-cellular spheroids and their use in stem cell culture. Current methods typically relying on the self-assembly of trypsinized, suspended stem cells, however, show limitations with respect to cell viability, throughput, and accurate recapitulation of the natural microenvironment. In this study, we developed a new system for engineering cell spheroids by self-assembly of micro-scale monolayer of stem cells. We prepared synthetic hydrogels with the surface of chemically formed micropatterns (squares/circles with width/diameter of 200 μm) on which mesenchymal stem cells isolated from human nasal turbinate tissue (hTMSCs) were selectively attached and formed a monolayer. The hydrogel is capable of thermally controlled expansion. As the temperature was decreased from 37 to 4 °C, the cell layer detached rapidly (10 min) and assembled to form spheroids with consistent size (∼100 μm) and high viability (90%). Spheroidization was significantly delayed and occurred with reduced efficiency on circle patterns compared to square patterns. Multi-physics mapping supported that delamination of the micro-scale monolayer may be affected by stress concentrated at the corners of the square pattern. In contrast, stress was distributed symmetrically along the boundary of the circle pattern. In addition, treatment of the micro-scale monolayer with a ROCK inhibitor significantly retarded spheroidization, highlighting the importance of contraction mediated by actin stress fibers for the stable generation of spheroidal stem cell structures. Spheroids prepared from the assembly of monolayers showed higher expression, both on the mRNA and protein levels, of ECM proteins (fibronectin and laminin) and stemness markers (Oct4, Sox2, and Nanog) compared to spheroids prepared from low-attachment plates, in which trypsinized single cells are assembled. The hTMSC spheroids also presented enhanced expression levels of markers related to tri-lineage (osteogenic, chondrogenic and adipogenic) differentiation. The changes in microcellular environments and functionalities were double-confirmed by using adipose derived mesenchymal stem cells (ADSCs). This spheroid engineering technique may have versatile applications in regenerative medicine for functionally improved 3D culture and therapeutic cell delivery.

Tendon-bone interface tissue is extremely challenging to engineer because it exhibits complex gradients of structure, composition, biologics, and cellular phenotypes. As a step toward engineering these transitional zones, we initially analyzed how different (topographical or biological) cues affect tenogenic differentiation of adipose-derived stem cells (ADSCs). We immobilized platelet-derived growth factor - BB (PDGF-BB) using polydopamine (PD) chemistry on random and aligned nanofibers and investigated ADSC proliferation and tenogenic differentiation. Immobilized PDGF greatly enhanced the proliferation and tenogenic differentiation of ADSCs; however, nanofiber alignment had no effect. Interestingly, the PDGF immobilized aligned nanofiber group showed a synergistic effect with maximum expression of tenogenic markers for 14 days. We also generated a nanofiber surface with spatially controlled presentation of immobilized PDGF on an aligned architecture, mimicking native tendon tissue. A gradient of immobilized PDGF was able to control the phenotypic differentiation of ADSCs into tenocytes in a spatially controlled manner, as confirmed by analysis of the expression of tenogenic markers and immunofluorescence staining. We further explored the gradient formation strategy by generation of a symmetrical gradient on the nanofiber surface for the generation of a structure mimicking bone-patellar-tendon-bone with provision for gradient immobilization of PDGF and controlled mineralization. Our study reveals that, together with biochemical cues, favorable topographical cues are important for tenogenic differentiation of ADSCs, and gradient presentation of PDGF can be used as a tool for engineering stem cell-based bone-tendon interface tissues.

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.

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.

Stem cells with mineralized materials have been used for bone regeneration; however, engineering the complex vascularized structure of the natural bone remains a challenge. Here, we developed platelet-derived growth factor (PDGF) and bio-mineral coated fibers which were then assembled with human adipose-derived stem cells (hADSCs) to form spheroids as building blocks for vascularized bone regeneration. The PDGF incorporated within the spheroid increased the proliferation of hADSCs, which was characterized by Ki-67 staining and DNA contents. Furthermore, the PDGF enhanced not only osteogenic differentiation, but also endothelial differentiation of hADSCs; the cells within the spheroids showed significantly greater gene expression by 2.46 ± 0.14 fold for osteocalcin (OCN) and by 12.85 ± 3.36 fold for von Willebrand factor (vWF) than those without PDGF. Finally, at two months following transplantation of PDGF-incorporated spheroids onto in vivo mouse calvarial defect, the regenerated bone area (42.48 ± 10.84%) was significantly enhanced and the greatest number of capillaries and arterioles with indication of transplanted hADSCs were observed. Moreover, millimeter-scale in vitro tissue prepared by fused assembly of the spheroids exhibited greater mRNA expression-associated to endothelial lineage. Taken together, these findings indicate that stem cell spheroids incorporating PDGF and bio-minerals could be used as a module for successful vascularized bone regeneration.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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 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.

Poly(lactic-co-glycolic acid)(PLGA)/biphasic calcium phosphate (BCP) composite nanofibers with different BCP to PLGA ratios were fabricated using the electrospinning technique. The scanning electron microscopy (SEM) images showed a similar morphology and fibers in all groups. The incorporated BCP was dispersed homogenously throughout the nanofibers, and the surface roughness was affected by the input amount of BCP. The increase in amount of BCP incorporated was confirmed by several methods. BCP incorporation into the PLGA nanofibers did not affect the initial adhesion of osteoblasts and their adherent morphology. However, the proliferation of the cells cultured on the composite nanofibers for 10 days with larger amounts of BCP was delayed, suggesting that incorporated BCP may facilitate the switch from proliferation to differentiation of the osteoblasts. The incorporation of BCP enhanced the expression of osteogenic genes, as well as induced calcium deposition by the osteoblasts in the extracellular matrix(ECM) after 21 days of culture on the PLGA/BCP composite nanofibers. Overall, these results can provide evidence of the potential of BCP incorporation into the biomaterials for effective bone regeneration.

In this study, novel fibrous matrices were developed as a depot to store and liberate growth factors in a controlled manner. Specifically, heparin was covalently conjugated onto the surface of fibrous matrices (composites of poly[caprolactone] and gelatin crosslinked with genipin), and basic fibroblast growth factor (bFGF) was then reversibly immobilized. The immobilization of bFGF was controlled as a function of the amount of conjugated heparin. The sustained release of bFGF from the fibrous matrices was successfully achieved over 4 weeks whereas physical adsorption of bFGF released quickly. The bFGF released from the fibrous matrices significantly enhanced in vitro proliferation of human umbilical vein endothelial cells. From the in vivo study, the group implanted with a higher amount of immobilized bFGF significantly facilitated neo-blood vessel formation as compared with other implantation groups. These results indicate that the sustained release of bFGF is important for the formation of blood vessels and that our fibrous matrices could be useful for regulation of tissue damage requiring angiogenesis. Further, our system can be combined with other growth factors with heparin binding domains, representing a facile depot for spatiotemporal control over the delivery of bioactive molecules in regenerative medicine.

In this study, we fabricated non-woven matrices using blends of polycaprolactone and gelatin with various spinning volumes to control the immobilized heparin content, which was ultimately intended to increase the immobilization efficiency of bFGF. The amount of bFGF on the heparin conjugated fibrous matrices depended on the thicknesses of the swollen matrices ranging from 35.4 ± 6.5 to 162.3 ± 14.0 ng and ≈90% of the bFGF was gradually released over a period of up to 56 d. The released bFGF enhanced the proliferation of human umbilical vein endothelial cells and human mesenchymal stem cells. In conclusion, our heparin-conjugated fibrous matrices have the potential to be used as a growth factor delivery system in tissue engineering applications.

Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Therefore, myotube formation plays an important role in restoring muscular functions, and substrates to promote the differentiation of myoblasts to myotubes need to be developed for muscle tissue engineering. In this study, we developed electrically conductive composite fibers of poly(L-lactide-co-epsilon-caprolactone) (PLCL) blended with polyaniline (PANi) using an electrospinning method, and then investigated the effect of these composite fibers on the differentiation of myoblasts. The prepared PLCL/PANi fibers showed no significant difference in fiber diameter or contact angle, regardless of the incorporation of PANi. The fibers containing 30% PANi (PLCL/PANi-30) maintained elastic properties of maximum elongation at break (160+/-14.4%). The composite fibers were cytocompatible, as the DNA content on each fiber was similar for up to 8 days of C2C12 myoblast culture. After 4 days of culture, the number of cells positive for sarcomeric myosin was 3.6-times greater on the electrically conductive fibers (21+/-1 and 19+/-2 for PLCL/PANi-15 and -30 fibers, respectively) than on the PLCL/PANi-0 fibers (6+/-2). Furthermore, the level of myogenin expression detected on day 8 of culture on PLCL/PANi-15 was approximately 1.6-fold greater than the PLCL/PANi-0 fibers. Similar results were observed for the expression of other genes including troponin T (2-fold greater) and the myosin heavy chain gene (3-fold greater). These results indicate that electrically conductive substrates can modulate the induction of myoblasts into myotube formation without additional electrical stimulation, suggesting that these fibers may have potential as a temporary substrate for skeletal tissue engineering.

Aims Cardiac tissue engineering has been proposed as an appropriate method to repair myocardial infarction (MI). Evidence suggests that a cell with scaffold combination was more effective than a cell-only implant. Nevertheless, to date, there has been no research into elastic biodegradable poly(lactide-co-e-caprolactone) (PLCL) scaffolds. The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) with elastic biodegradable PLCL scaffold transplants in a rat MI model. Methods and results Ten days after inducing MI through the cryoinjury method, a saline control, MSC, PLCL scaffold, or MSC-seeded PLCL scaffold was transplanted onto the hearts. Four weeks after transplantation, cardiac function and histology were evaluated. Transplanted MSCs survived and differentiated into cardiomyocytes in the injured region. Left ventricular ejection fraction in the MSC + PLCL group increased by 23% compared with that in the saline group; it was also higher in the MSC group. The infarct area in the MSC + PLCL group was decreased by 29% compared with that in the saline group; it was also reduced in the MSC group. Conclusion Mesenchymal stem cells plus PLCL should be an excellent combination for cardiac tissue engineering.