Mussel Adhesion-Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose-Derived Stem Cells.

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.

Effective gene delivery into human stem cells with a cell-targeting Peptide-modified bioreducible polymer.

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.

Materials from Mussel-Inspired Chemistry for Cell and Tissue Engineering Applications.

Current advances in biomaterial fabrication techniques have broadened their application in different realms of biomedical engineering, spanning from drug delivery to tissue engineering. The success of biomaterials depends highly on the ability to modulate cell and tissue responses, including cell adhesion, as well as induction of repair and immune processes. Thus, most recent approaches in the field have concentrated on functionalizing biomaterials with different biomolecules intended to evoke cell- and tissue-specific reactions. Marine mussels produce mussel adhesive proteins (MAPs), which help them strongly attach to different surfaces, even under wet conditions in the ocean. Inspired by mussel adhesiveness, scientists discovered that dopamine undergoes self-polymerization at alkaline conditions. This reaction provides a universal coating for metals, polymers, and ceramics, regardless of their chemical and physical properties. Furthermore, this polymerized layer is enriched with catechol groups that enable immobilization of primary amine or thiol-based biomolecules via a simple dipping process. Herein, this review explores the versatile surface modification techniques that have recently been exploited in tissue engineering and summarizes polydopamine polymerization mechanisms, coating process parameters, and effects on substrate properties. A brief discussion of polydopamine-based reactions in the context of engineering various tissue types, including bone, blood vessels, cartilage, nerves, and muscle, is also provided.

Volume-stable adipose tissue formation by implantation of human adipose-derived stromal cells using solid free-form fabrication-based polymer scaffolds.

Regeneration of volume-stable adipose tissue is required for treatment of soft-tissue loss due to cancer, trauma, burns and for correctional cosmetic surgery. In this study, we hypothesized that transplantation of human adipose-derived stromal cells (hADSCs) using polycaprolactone (PCL) scaffolds fabricated with a solid free-form fabrication method would better maintain the volume of regenerated adipose tissues, as compared with the use of fibrin gel. Six weeks after implantation into the dorsal subcutaneous pockets of athymic mice, the volumes and adipose tissue areas of hADSC-PCL scaffold implants were significantly larger than those of hADSC-fibrin implants. In addition, the mRNA expression of adipogenic genes was more extensive in the hADSC-PCL scaffold implants.

Mussel-inspired immobilization of vascular endothelial growth factor (VEGF) for enhanced endothelialization of vascular grafts.

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

Sulfobetaine polymers for effective permeability into multicellular tumor spheroids (MCTSs).

Multicellular tumor spheroids (MCTSs) are attractive for drug screening before animal tests because they emulate an in vivo microenvironment. The permeability of the MCTSs and tumor tissues towards the candidate drugs is not sufficient even though the drugs can penetrate monolayer cultured cells; therefore, nanocarriers are required to enhance permeability and deliver drugs. In this study, we prepared zwitterionic polymers of sulfobetaine methacrylates and (meth)acrylamides with or without hydroxy groups between the zwitterions to serve as highly permeable nanocarriers. In the sulfobetaine polymers, poly(2-hydroxy-3-((3-methacrylamidopropyl)dimethylammonio)propane-1-sulfonate), P(OH-MAAmSB), the hydroxy group containing methacrylamide polymer exhibited little cytotoxicity and membrane translocation ability against monolayer cultured cells. Moreover, the excellent permeability of the hepatocyte MCTS enabled P(OH-MAAmSB) to permeate it and reach the center region (∼325 µm in diameter) at approximately 150 s, although poly(trimethyl-2-methacroyloxyethylammonium), a cationic polymer, penetrated just 1 to 2 layers from the periphery. The superior permeability of P(OH-MAAmSB) might be due to its good solubility and side chain conformation. P(OH-MAAmSB) is a promising nanocarrier with membrane translocation and permeability.

Tailored polyethylene glycol grafting on porous nanoparticles for enhanced targeting and intracellular siRNA delivery.

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.

Evaluation of the anti-oxidative and ROS scavenging properties of biomaterials coated with epigallocatechin gallate for tissue engineering.

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.

On/off switchable physical stimuli regulate the future direction of adherent cellular fate.

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.

In situ forming hydrogels based on tyramine conjugated 4-Arm-PPO-PEO via enzymatic oxidative reaction.

Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical applications like implants, drugs and cell delivery carriers because of their high biocompatibility, high water contents and excellent permeability for nutrients and metabolites. Especially, in situ forming hydrogel systems have received much attention because of their easy application based on minimal invasive techniques. Chemical cross-linking systems fabricated using enzymatic reactions have various advantages, such as high biocompatibility and easy control of reaction rates under mild condition. In this study, we report enzyme-triggered injectable and biodegradable hydrogels composed of Tetronic-tyramine conjugates. The Tetronic-tyramine conjugates were synthesized by first reacting Tetronic with succinic anhydride and subsequent conjugation with tyramine using DCC/NHS as coupling reagents. The chemical structure of Tetronic-succinic anhydride-tyramine (Tet-SA-TA) copolymer was characterized by (1)H NMR and FTIR. The hydrogels were prepared from a Tet-SA-TA solution above 3 wt % in the presence of horseradish peroxidase (HRP) and H(2)O(2) under physiological conditions. Their mechanical property, gelation time, swelling ratio and degradation time were evaluated at different polymer, HRP, and H(2)O(2) concentrations. In addition, a cyto-compatibility study was performed using the MC3T3-E1 cell line. In the cytotoxicity test, it was clear that the Tet-SA-TA hydrogel had no apparent cytotoxicity except for the hydrogel formed with 0.25 wt % H(2)O(2) due to the cytotoxicity of residual H(2)O(2). In conclusion, the obtained results demonstrated that the Tet-SA-TA hydrogel has great potential for use as an injectable scaffold for tissue engineering and as a drug carrier for controlled drug delivery systems.

Preparation and characterization of CdSe/ZnS quantum dots encapsulated in poly(ethylene glycol)-b-poly(D,L-lactide) micelle nanoparticles.

The final goal of this study is to develop multi-functional organic/inorganic hybrid nanoparticles, which can be utilized as biomedical imaging probes and drug delivery carriers. As an initial step toward this goal, we encapsulated CdSe/ZnS quantum dots (QDs) into poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) micelles using a solid dispersion method. The size and fluorescent intensity of QDs encapsulated in PEG-PLA micelles depended on the amount of incorporated QDs. For example, when the amount of QDs increased from 0.1 to 1.0 microg, the mean diameter increased from 24.2 +/- 6.0 to 211.2 +/- 6.5 nm and the fluorescent intensity changed from 10.2 +/- 1.0 to 469.9 +/- 15.6 (RFU). Stability studies showed that the size and zeta-potential (ZP) of QDs encapsulated in PEG-PLA micelles (QEMs) did not change significantly in response to a change in pH conditions or under a 10% serum condition. We also tested the cytotoxicity and cellular uptake of the QEMs. The viability of HeLa cells treated with micelles for 24 h was 80-100% in various concentration ranges of micelles. Confocal laser scanning microscopic images showed that the QEMs penetrated into the cells, particularly into the cytosolic compartments. Our results suggest that the QEMs may be a promising multi-functional nanocarrier for biomedical imaging and drug delivery.

Polydopamine-assisted one-step modification of nanofiber surfaces with adenosine to tune the osteogenic differentiation of mesenchymal stem cells and the maturation of osteoclasts.

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.

Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering.

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.

Surface engineering of titanium alloy using metal-polyphenol network coating with magnesium ions for improved osseointegration.

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).

Fabrication of size-controllable human mesenchymal stromal cell spheroids from micro-scaled cell sheets.

Recently, stromal cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stromal cell spheroids in different sizes from micro-scaled cell sheets (µCS) using thermosensitive hydrogels and investigated their effects on stromal cell function. Mesenchymal stromal 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 × 104 cells cm-2, respectively, on 400 µm micro-pattern). In addition, the spheroids were successfully fabricated regardless of stromal 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 were increased (13.2 ± 1.4, 325 ± 83.4 and 534.3 ± 121.5 pg ng-1 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 d 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.

Current Advances in Immunomodulatory Biomaterials for Bone Regeneration.

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.

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration.

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.

Hydrogels with an embossed surface: An all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids.

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 ±â€¯6 µm to 181 ±â€¯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.

Modulation of spreading, proliferation, and differentiation of human mesenchymal stem cells on gelatin-immobilized poly(L-lactide-co--caprolactone) substrates.

Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.

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

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 ±â€¯2.83 and 22.36 ±â€¯2.18 fold increase), OPN (14.24 ±â€¯1.71 and 15.076 ±â€¯1.38 fold increase), and OCN (4.36 ±â€¯0.41 and 5.63 ±â€¯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.