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Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. STATEMENT OF SIGNIFICANCE: Recently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.

Scaffolds functionalized with biomolecules have been developed for bone regeneration but inducing the regeneration of complex structured bone with neovessels remains a challenge. For this study, we developed three-dimensional printed scaffolds with bioactive surfaces coated with minerals and platelet-derived growth factor. The minerals were homogeneously deposited on the surface of the scaffold using 0.01 M NaHCO

Surface functionalization of nanoparticles with polyethylene glycol (PEG) has been widely demonstrated as an anti-opsonization strategy to reduce protein corona formation which is one of the major concerns affecting target receptor recognition. However, excessive surface passivation with PEG can lead to the strong inhibition of cellular uptake and less efficient binding to target receptors, resulting in reduced potential of targeted delivery. To improve specific cell targeting while reducing the nonspecific protein adsorption, a secondary packaging of the nanoparticles with shorter PEG chains, making the targeting ligands densely stretched out for enhanced molecular recognition is demonstrated. Particularly, we report the tailored surface functionalization of the porous nanoparticles that require the stealth shielding onto the open-pore region. This study shows that, in addition to the surface chemistry, the conformation of the PEG layers controls the cellular interaction of nanoparticles. Since the distance between neighboring PEG chains determines the structural conformation of the grafted PEG molecules, tailored PEG combinations can efficiently resist the adsorption of serum proteins onto the pores by transitioning the conformation of the PEG chains, thus significantly enhance the targeting efficiency (5-fold). The stretched brush PEG conformation with secondary packaging of shorter PEG chains could be a promising anti-opsonization and active targeting strategy for efficient intracellular delivery of nanoparticles.

Hydroxy group containing methacrylamide polymers exhibited excellent permeability into the hepatocyte multicellular tumor spheroids.

Techniques for harvest and delivery of cell sheets have been improving for decades. However, cell sheets with complicated patterns closely related to natural tissue architecture were hardly achieved. Here, we developed an efficient method to culture and harvest cell sheets with complex shape (noted as microtissues) using temperature-responsive hydrogel consisting of expandable polyethylene oxide polymer at low temperature. Firstly, a temperature-responsive hydrogel surface with honeycomb patterns (50 and 100 µm in width) were developed through microcontact printing of polydopamine (PD). The human dermal fibroblasts (HDFBs) and human umbilical vein endothelial cells (HUVECs) spontaneously formed honeycomb-shaped microtissues on the patterned hydrogel surface. The microtissues on the hydrogel were able to be harvested and directly delivered to the desired target through thermal expansion of the hydrogel at 4 °C with an efficiency close to 80% within 10 min which is faster than conventional method based on poly(N-isopropylacrylamide). The microtissues maintained their original honeycomb network and intact structures. Honeycomb-patterned cell sheets also were fabricated through serial seeding of various cell lines, including HDFBs, HUVECs, and human adipose-derived stem cells, in which cells were attached along the honeycomb pattern. The underlying honeycomb patterns in the cell sheets were successfully maintained for 3 days, even after delivery. In addition, patterned cell sheets were successfully delivered in vivo while maintaining an intact structure for 7 days. Together, our findings demonstrate that micropatterned temperature-responsive hydrogel is an efficient method of one-step culturing and delivery of complex microtissues and should prove useful in various tissue engineering applications. Statement of significance Scaffold-free cell delivery techniques, including cell sheet engineering, have been developed for decades. However, there is limited research regarding culture and delivery of microtissues with complex architecture mimicking natural tissue. Herein, we developed a micro-patterned hydrogel platform for the culture and delivery of honeycomb-shaped microtissues. Honeycomb patterns were chemically engineered on the temperature-responsive hydrogel through microcontact printing of polydopamine to selectively allow for human dermal fibroblast or human umbilical vein endothelial cell adhesion. They spontaneously formed honeycomb-shaped microtissues within 24 hr upon cell seeding and directly delivered to various target area including in vivo via thermal expansion of the hydrogel at 4 °C, suggesting that the micro-patterned hydrogel can be an efficient tool for culture and delivery of complex microtissue.

Multicellular spheroids are formed by strong cell-cell and cell-extracellular matrix interactions and are widely utilized in tissue engineering for therapeutic treatments or ex vivo tissue modeling. However, diffusion of oxygen into the spheroid gradually decreases, forming a necrotic core. In this study, polycaprolactone (PCL) fibers with pores and epigallocatechin gallate (EGCG) coating on their surface to provide a structural framework within the spheroids and investigated their ability to mitigate diffusional limitation and control over the proliferation of human adipose-derived stem cells (hADSCs) is engineered. The DNA content of composite spheroids prepared from fibers and hADSCs decreased in unadjusted cells (1224 ± 134 ng), in those with fibers with a smooth surface (SF) (1447 ± 331 ng), and in those EGCG-coated with SF (E-SF) (1437 ± 289 ng). Cells with fibers with pores on the surface (PF) (2020 ± 32 ng) and those with EGCG-coated PF (E-PF) (1911 ± 80 ng) increased after 7 days of culture, with a significantly greater number of proliferating cells (29 ± 8% and 30 ± 8%, respectively). These results indicate that physical modification through the formation of pores on the fiber surface alleviates diffusion limitation of composite spheroids, playing a dominant role over chemical modification.

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.

The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.

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.

Numerous methods have been introduced to produce 3D cell cultures that can reduce the need for animal experimentation. This study presents a unique 3D culture platform that features bioinspired strands of electrospun nanofibers (BSeNs) and aquatic cell lines to compensate for shortcomings in the current cell spheroid generation techniques. The use of BSeNs in 3D zebrafish liver cell cultures is found to improve liver and reproductive functions through spheroid-based in vitro assays such as whole transcriptome sequencing and reproductive toxicity testing, with optimized properties exhibiting results similar to those obtained for fish embryo acute toxicity (FET, OECD TG 236) following exposure to environmental endocrine-disrupting chemicals (17β-Estradiol (E2), 4-hydroxytamoxifen (4-HT), and bisphenol compounds (bisphenol A (BPA) and 9,9-Bis(4-hydroxyphenyl)fluorene (BPFL)). These findings indicate that the beneficial effects of bioinspired materials that closely mimic ECM environments can yield efficient zebrafish cells with intrinsic functions and xenobiotic metabolism similar to those of zebrafish embryos. As a closer analog for the in vivo conditions that are associated with exposure to potentially hazardous chemicals, the straightforward culture model introduced in this study shows promise as an alternative tool that can be used to further eco-environmental assessment.

Although titanium-based implants are widely used in orthopedic and dental clinics, improved osseointegration at the bone-implant interface is still required. In this study, we developed a titanium alloy (Ti-6Al-4V, Ti) coated with epigallocatechin gallate (EGCG) and magnesium ions (Mg2+) in a metal-polyphenol network (MPN) formation. Specifically, Ti discs were coated with EGCG in MgCl2 by controlling their concentrations and pH, with the amount of coating increasing with the coating time. An in vitro culture of human adipose-derived stem cells (hADSCs) on the EGCG-Mg2+-coated Ti showed significantly enhanced ALP activity and mRNA expression of osteogenic markers. In addition, the EGCG-Mg2+-coated Ti enhanced the mineralization of hADSCs, significantly increasing the calcium content (22.2 ± 5.0 μg) compared with cells grown on Ti (13.5 ± 0.3 μg). Treatment with 2-APB, an inhibitor of Mg2+ signaling, confirmed that the enhancement of osteogenic differentiation in the hADSCs was caused by the synergistic influence of EGCG and Mg2+. The EGCG-Mg2+ coating significantly reduced the osteoclastic maturation of Raw264.7 cells, reducing tartrate-resistant acid phosphatase activity (5.4 ± 0.4) compared with that of cells grown on Ti (1.0 ± 0.5). When we placed Ti implants onto rabbit tibias, the bone-implant contact (%) was greater on the EGCG-Mg2+-coated Ti implants (8.1 ± 4.3) than on the uncoated implants (4.4 ± 2.0). Therefore, our MPN coating could be a reliable surface modification for orthopedic implants to enable the delivery of an osteoinductive metal ion (Mg2+) with the synergistic benefits of a polyphenol (EGCG).

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.

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.

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.

Sodium alginate (SA) is a non-toxic, biocompatible and biodegradable natural polymer. It has good printability, which is why it is often used in inks for three-dimensional (3D) printing in tissue engineering fields. However, pure SA hydrogel has unstable mechanical properties. In this study, we fabricated a structurally stable 3D scaffold by blending SA with poly(vinyl alcohol) (PVA) to enhance the mechanical properties of the scaffold. Moreover, we characterized the surface morphology and various mechanical properties of the scaffolds using scanning electron microscopy, Fourier transform-infrared spectroscopy, thermogravimetric analysis and several mechanical tests. The SA/PVA scaffolds were mechanically more stable than the pure SA scaffold and are good candidate as implantable biodegradable scaffolds for tissue engineering and medical applications.

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 .

Cell spheroid culture can be an effective approach for providing an engineered microenvironment similar to an in vivo environment. Our group had recently developed a method for harvesting uniformly sized multicellular spheroids via self-assembly of micro-scaled cell sheets (μCSs) induced by the expansion of temperature-sensitive hydrogels. However, the μCS assembly process was not fully understood. In this study, we investigated the effects of cell number, pattern shape, and contractile force of cells on spheroid formation from micropatterned (width of square pattern from 100-300 μm) hydrogels. We used human dermal fibroblasts (HDFBs) as a model cell type and cultured them for 24 and 72 h. The self-assembly of μCSs cultured on square micropatterns for 72 h rapidly occurred within 4 min after reducing the temperature from 37 to 4 °C. In addition, the size distribution of spheroids was narrower with μCSs from a 72 h culture. Treatment with a ROCK1 inhibitor disrupted cytoskeletal actin fibers and the corresponding μCSs were not detached from the hydrogel. The assembly of the μCS was also affected by the micropattern shape, and the spheroid harvest efficiency was decreased to 60% when using a circular micropattern, which was explained by the stress direction on the circular versus square micropattern upon hydrogel expansion. Therefore, we confirmed that the factors controlling cell-cell interactions are important for spheroid formation using micropatterned hydrogel systems. Finally, the μCSs with dual layers of HDFBs labeled with DiD and DiO dyes resulted in the formation of spheroids with discretely localized colors within the core and shell, respectively, which suggests an outside-in assembly of detached μCSs. In consideration of these complex environmental factors, our system could be utilized in various applications as a three-dimensional culture system to fabricate cell spheroids.

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.

Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity compared to individual cells in suspension. However, there is limited information about the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into Matrigel for investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in Matrigel, denoted as sandwich-culture, with specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we sandwich-cultured human umbilical vein endothelial cells (HUVECs) and ASC in co-cultured spheroids in Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when inter-spheroid distance was 200 µm. Similar results were obtained from in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in hydrogel.

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.

Substrates modified with gradient surface chemistry are of fundamental importance for designing a new bio-interface in biomaterial research and tissue engineering. However, current gradient fabrication strategies are not easily accessible to most laboratories due to complex, expensive, and expertise-requiring procedures. In this study, we generated a gradient of polydopamine (PD) coating on a PLLA nanofiber surface using a spatially restricted supply of oxygen in the reaction solution. Analysis of the oxygen distribution revealed that oxygen availability varied along different reaction solution depths during dopamine polymerization. We then extensively investigated the effects of different parameters, such as tilting angle, reaction time, pH of the reaction solution, and concentration of dopamine, on PD gradient formation, which should be appropriately modulated for PD gradient on nanofibers. Further, culturing of human mesenchymal stem cells (hMSCs) on the PD gradient nanofiber resulted in a gradient of adhesion and spreading from high to low PD coating. However, the proliferation rate was not affected by the PD gradient, with an approximately 3-fold change after 5 days of culture. Maintenance of the stem cell density gradient on the PD gradient nanofiber resulted in controlled osteogenic differentiation, which was greater in the higher PD-coated area. Interestingly, stemness analysis showed a reverse trend relative to osteogenic differentiation of hMSCs. In summary, the spatially controlled polymerization of dopamine can be a versatile tool to generate substrates with gradient surface chemistry, which holds promise to direct stem cell behavior.

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 (

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

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.

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.

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.

Biological responses on biomaterials occur either on their surface or at the interface. Therefore, surface characterization is an essential step in the fabrication of ideal biomaterials for achieving effective control of the interaction between the material surface and the biological environment. Herein, we applied femtosecond laser ablation on electrospun fibrous scaffolds to fabricate various hierarchical patterns with a focus on the alignment of cells. We investigated the simultaneously stimulated response of cardiomyoblasts based on multiple topographical cues, including scales, oriented directions, and spatial arrangements, in the fibrous scaffolds. Our results demonstrated a synergistic effect on cell behaviors of one or more structural arrangements in a homogeneous orientation, whereas antagonistic effects were observed for cells arranged on a surface with heterogeneous directions. Taken together, these results indicate that our hierarchically patterned fibrous scaffolds may be useful tools for understanding the cellular behavior on fibrous scaffolds used to mimic an extracellular matrix-like environment. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1732-1742, 2018.