Development and evaluation of an injectable ChitHCl-MgSO4-DDA hydrogel for bone regeneration: In vitro and in vivo studies on cell migration and osteogenesis enhancement.

Nonunion and delayed union of the bone are situations in orthopedic surgery that can occur even if the bone alignment is correct and there is sufficient mechanical stability. Surgeons usually apply artificial bone grafts in bone fracture gaps or in bone defect sites for osteogenesis to improve bone healing; however, these bone graft materials have no osteoinductive or osteogenic properties, and fit the morphology of the fracture gap with difficulty. In this study, we developed an injectable chitosan-based hydrogel with MgSO4 and dextran oxidative, with the purpose to improve bone healing through introducing an engineered chitosan-based hydrogel. The developed hydrogel can gelate and fit with any morphology or shape, has good biocompatibility, can enhance the cell-migration capacity, and can improve extracellular calcium deposition. Moreover, the amount of new bone formed by injecting the hydrogel in the bone tunnel was assessed by an in vivo test. We believe this injectable chitosan-based hydrogel has great potential for application in the orthopedic field to improve fracture gap healing.

Injectable ChitHCl-DDA tissue adhesive with high adhesive strength and biocompatibility for torn meniscus repair and regeneration.

Suture pull-through is a clinical problem in meniscus repair surgery due to the sharp leading edge of sutures. Several tissue adhesives have been developed as an alternative to traditional suturing; however, there is still no suitable tissue adhesive specific for meniscus repair treatment due to unsatisfactory biosafety, biodegradable, sterilizable, and tissue-bonding characteristics. In this study, we used a tissue adhesive composed of chitosan hydrochloride reacted with oxidative periodate-oxidized dextran (ChitHCl-DDA) combined with a chitosan-based hydrogel and oxidative dextran to attach to the meniscus. We conducted viscoelastic tests, viscosity tests, lap shear stress tests, Fourier transform infrared (FTIR) spectroscopy, swelling ratio tests, and degradation behavior tests to characterize these materials. An MTT assay, alcian blue staining, migration assay, cell behavior observations, and protein expression tests were used to understand cell viability and responses. Moreover, ex vivo and in vivo tests were used to analyze tissue regeneration and biocompatibility of the ChitHCl-DDA tissue adhesive. Our results revealed that the ChitHCl-DDA tissue adhesive provided excellent tissue adhesive strength, cell viability, and cell responses. This tissue adhesive has great potential for torn meniscus tissue repair and regeneration.

Diabetes Primes Neutrophils for Neutrophil Extracellular Trap Formation through Trained Immunity.

Neutrophils are primed for neutrophil extracellular trap (NET) formation during diabetes, and excessive NET formation from primed neutrophils compromises wound healing in patients with diabetes. Here, we demonstrate that trained immunity mediates diabetes-induced NET priming in neutrophils. Under diabetic conditions, neutrophils exhibit robust metabolic reprogramming comprising enhanced glycolysis via the pentose phosphate pathway and fatty acid oxidation, which result in the accumulation of acetyl-coenzyme A. Adenosine 5'-triphosphate-citrate lyase-mediated accumulation of acetyl-coenzyme A and histone acetyltransferases further induce the acetylation of lysine residues on histone 3 (AcH3K9, AcH3K14, and AcH3K27) and histone 4 (AcH4K8). The pharmacological inhibition of adenosine 5'-triphosphate-citrate lyase and histone acetyltransferases completely inhibited high-glucose-induced NET priming. The trained immunity of neutrophils was further confirmed in neutrophils isolated from patients with diabetes. Our findings suggest that trained immunity mediates functional changes in neutrophils in diabetic environments, and targeting neutrophil-trained immunity may be a potential therapeutic target for controlling inflammatory complications of diabetes.

Extracellular vesicles from dHL-60 cells as delivery vehicles for diverse therapeutics.

Extracellular vesicles (EVs) are membrane-derived heterogeneous vesicles that mediate intercellular communications. They have recently been considered as ideal vehicles for drug-delivery systems, and immune cells are suggested as a potential source for drug-loaded EVs. In this study, we investigated the possibility of neutrophils as a source for drug-loaded EVs. Neutrophil-like differentiated human promyelocytic leukemia cells (dHL-60) produced massive amounts of EVs within 1 h. The dHL-60 cells are also easily loaded with various cargoes such as antibiotics (penicillin), anticancer drug (paclitaxel), chemoattractant (MCP-1), miRNA, and Cas9. The EVs derived from the dHL-60 cells showed efficient incorporation of these cargoes and significant effector functions, such as bactericidal activity, monocyte chemotaxis, and macrophage polarization. Our results suggest that neutrophils or neutrophil-like promyelocytic cells could be an attractive source for drug-delivery EVs.

Neutrophil-derived trail is a proinflammatory subtype of neutrophil-derived extracellular vesicles.

Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo. Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV.

Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.

Despite great progress in biomaterial design strategies for replacing damaged articular cartilage, prevention of stem cell-derived chondrocyte hypertrophy and resulting inferior tissue formation is still a critical challenge. Here, by using engineered biomaterials and a high-throughput system for screening of combinatorial cues in cartilage microenvironments, we demonstrate that biomaterial cross-linking density that regulates matrix degradation and stiffness-together with defined presentation of growth factors, mechanical stimulation, and arginine-glycine-aspartic acid (RGD) peptides-can guide human mesenchymal stem cell (hMSC) differentiation into articular or hypertrophic cartilage phenotypes. Faster-degrading, soft matrices promoted articular cartilage tissue formation of hMSCs by inducing their proliferation and maturation, while slower-degrading, stiff matrices promoted cells to differentiate into hypertrophic chondrocytes through Yes-associated protein (YAP)-dependent mechanotransduction. in vitro and in vivo chondrogenesis studies also suggest that down-regulation of the Wingless and INT-1 (WNT) signaling pathway is required for better quality articular cartilage-like tissue production.

Multi-peptide presentation and hydrogel mechanics jointly enhance therapeutic duo-potential of entrapped stromal cells.

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Comparison of cytotoxicity of black phosphorus nanosheets in different types of fibroblasts.

BACKGROUND: Two-dimensional black phosphorus nanosheets (BPNSs) have recently emerged as a successive novel nanomaterial owing to their uniqueness in optical and electrical properties. Although BPNSs have found a wide range of biomedical applications, their biosafety is still a major concern to be addressed. METHODS: In this study, we have prepared layered BPNSs using liquid exfoliation procedure, and evaluated their physicochemical properties using Fourier Transform-infrared (FTIR) spectroscopy, Raman spectroscopy, atomic force microscopy, and Zetasizer analyses. We have investigated potential cytotoxicity of BPNSs against three different types of fibroblast cells, i.e. mouse embryonic fibroblast (NIH3T3), primary cultured normal human dermal fibroblast (nHDF), and fibrosarcoma (HT1080). Cell counting kit-8 (CCK-8) assay was carried out to assess cellular metabolic activity in cells whereas lactate dehydrogenase (LDH) activity assay was helpful to study plasma membrane integrity. RESULTS: Our salient research findings showed that BPNSs were polydispersed in solution due to aggregation. Toxic response of BPNSs against fibroblast cells was in the order, HT1080>nHDF>NIH3T3. The nanosheets reduced the number of cancerous cells with significant difference to normal cells. CONCLUSIONS: We suggest that BPNSs can be considered for cancer treatment as they destroy cancerous cells effectively. However, a comprehensive study is required to elucidate other biological effects of BPNSs.

Fabrication of core-shell spheroids as building blocks for engineering 3D complex vascularized tissue.

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.

Toxicity of Zero- and One-Dimensional Carbon Nanomaterials.

The zero (0-D) and one-dimensional (1-D) carbon nanomaterials have gained attention among researchers because they exhibit a larger surface area to volume ratio, and a smaller size. Furthermore, carbon is ubiquitously present in all living organisms. However, toxicity is a major concern while utilizing carbon nanomaterials for biomedical applications such as drug delivery, biosensing, and tissue regeneration. In the present review, we have summarized some of the recent findings of cellular and animal level toxicity studies of 0-D (carbon quantum dot, graphene quantum dot, nanodiamond, and carbon black) and 1-D (single-walled and multi-walled carbon nanotubes) carbon nanomaterials. The in vitro toxicity of carbon nanomaterials was exemplified in normal and cancer cell lines including fibroblasts, osteoblasts, macrophages, epithelial and endothelial cells of different sources. Similarly, the in vivo studies were illustrated in several animal species such as rats, mice, zebrafish, planktons and, guinea pigs, at various concentrations, route of administrations and exposure of nanoparticles. In addition, we have described the unique properties and commercial usage, as well as the similarities and differences among the nanoparticles. The aim of the current review is not only to signify the importance of studying the toxicity of 0-D and 1-D carbon nanomaterials, but also to emphasize the perspectives, future challenges and possible directions in the field.

Predictors and impact of hemorrhagic transformations after endovascular thrombectomy in patients with acute large vessel occlusions.

BACKGROUND: Predictors and impact of hemorrhagic transformation (HT) after thrombectomy remain to be elucidated. OBJECTIVE: To investigate the independent predictors and impact of each hemorrhagic infarction (HI) and parenchymal hematoma (PH) after thrombectomy in patients with acute stroke due to intracranial large vessel occlusion (LVO). MATERIALS AND METHODS: We retrospectively reviewed data from 400 patients with acute LVO who underwent thrombectomy. Logistic regression analyses were performed to determine independent predictors of HI and PH on post-treatment CT scans. Associations between HT and poor outcome (modified Rankin Scalescore ≥3) at 90 days were analyzed. RESULTS: HT was observed in 98 patients (62 HIs (15.5%) and 36 PHs (9%)). Independent predictors of HI were male sex, atrial fibrillation, and time from symptom onset to groin puncture. Hyperlipidemia (OR=0.221, 95% CI 0.064 to 0.767, P=0.017) and successful reperfusion (OR=0.246, 95% CI 0.093 to 0.651, P=0.005) were independently associated with a lower chance of PH, while hypertension (OR=2.260, 95% CI 1.014 to 5.035, P=0.046) and longer procedure duration (OR=1.046, 95% CI 1.016 to 1.077, P=0.003) were independently associated with a higher chance of PH. Only PH (OR=10.154, 95% CI 3.260 to 31.632, P<0.001) was an independent predictor of poor outcome. CONCLUSIONS: PH is independently associated with poor outcome, whereas HI does not predict outcome after thrombectomy in patients with acute LVO. Our findings suggest that rapid and successful reperfusion is essential to prevent PH in patients undergoing thrombectomy for acute LVO. In addition, our study suggests that hyperlipidemia is associated with a lower risk of PH in such patients.

Dual specificity phosphatase DUSP6 promotes endothelial inflammation through inducible expression of ICAM-1.

Tumor necrosis factor (TNF)-α activates a diverse array of signaling pathways in vascular endothelial cells (ECs), leading to the inflammatory phenotype that contributes to the vascular dysfunction and neutrophil emigration in patients with sepsis. To date, it is not well understood what key regulator might coordinate signaling pathways to achieve inflammatory response in TNF-α-stimulated ECs. This study investigated the role of dual specificity phosphatase-6 (DUSP6) in the regulation of endothelial inflammation. Using knockout mice, we found that DUSP6 is important for TNF-α-induced endothelial intercellular adhesion molecule-1 (ICAM-1) expression in aorta and in vein. Moreover, genetic deletion of Dusp6 in pulmonary circulation significantly alleviated the susceptibility of mice to lung injury caused by neutrophil recruitment during experimental sepsis induced by TNF-α or lipopolysaccharide (LPS). The role of DUSP6 was further investigated in primary human umbilical vein endothelial cells (HUVECs). Employing RNAi approach in which endogenous DUSP6 was ablated, we showed a critical function of DUSP6 to facilitate TNF-α-induced ICAM-1 expression and endothelial leukocyte interaction. Interestingly, DUSP6-promoted endothelial inflammation is independent of extracellular signaling-regulated kinase (ERK) signaling. On the other hand, inducible DUSP6 leads to activation of canonical nuclear factor (NF)-κB-mediated transcription of ICAM-1 gene in TNF-α-stimulated human ECs. These results are the first to demonstrate a positive role of DUSP6 in endothelial inflammation-mediated pathological process and the underlying mechanism through which DUSP6 promotes NF-κB signaling in the inflamed ECs. Our findings suggest that manipulation of DUSP6 holds great potential for the treatment of acute inflammatory diseases.

Engineering spheroids potentiating cell-cell and cell-ECM interactions by self-assembly of stem cell microlayer.

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.

Graded functionalization of biomaterial surfaces using mussel-inspired adhesive coating of polydopamine.

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

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis.

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication.

Transfer stamping of human mesenchymal stem cell patches using thermally expandable hydrogels with tunable cell-adhesive properties.

Development of stem cell delivery system with ability of control over mutilineage differentiation and improved engraft efficiency is imperative in regenerative medicine. We herein report transfer stamping of human mesenchymal stem cells (hMSCs) patches using thermally expandable hydrogels with tunable cell-adhesive properties. The hydrogels were prepared from functionalized four arm copolymer of Tetronic(®), and the cell adhesion on the hydrogel was modulated by incorporation of fibronectin (FN) or cell-adhesive peptide (RGD). The resulting hydrogels showed spontaneous expansion in size within 10 min in response to the temperature reduction from 37 to 4°C. The adhesion and proliferation of hMSCs on FN-hydrogels were positively tunable in proportion to the amount of FN within hydrogels with complete monolayer of hMSCs (hMSC patch) being successfully achieved. The hMSC patch on the hydrogel was faced to the target substrate, which was then easily detached and re-attached to the target when the temperature was reduced from 37°C up to 4°C. We found that the transfer stamping of cell patch was facilitated at lower temperature of 4°C relative to 25°C, with the use of thinner hydrogels (0.5 mm in thickness relatively to 1.0 or 1.5 mm) and longer transfer time (>15 min). Notably, the hMSC patch was simply transferred from the hydrogel to the subcutaneous mouse skin tissue within 15 min with cold saline solution being dropped to the hydrogel. The hMSC patch following osteogenic or adipogenic commitment was also achieved with long-term culture of hMSCs on the hydrogel, which was successfully detached to the target surface. These results suggest that the hydrogels with thermally expandable and tunable cell-adhesive properties may serve as a universal substrate to harvest hMSC patch in a reliable and effective manner, which could potentially be utilized in many cell-sheet based therapeutic applications.

Rapid transfer of endothelial cell sheet using a thermosensitive hydrogel and its effect on therapeutic angiogenesis.

In this study, thermosensitive hydrogels incorporated with multiple cell-interactive factors were developed as a substrate to form monolayer of human umbilical vein endothelial cells (HUVECs) that can be detached and transferrable to target sites as a cell-sheet in response to temperature change. The cell adhesive peptide (RGD) and growth factor (bFGF) covalently incorporated within the hydrogel significantly enhanced adhesion and proliferation of HUVECs, allowing for the formation of their confluent monolayer. Meanwhile, the precisely controllable change in the size of the hydrogels was observed by a repeated increase and decrease in temperature from 37 to 4 °C. By exploiting this unique behavior, the detachment and transfer of HUVEC sheet confluently cultured at 37 °C was rapidly induced within 10 min by expansion of the hydrogels when the temperature was decreased to 4 °C. The transferred cell sheet was highly viable and maintained robust cell-cell junction. Finally, the process of cell sheet transfer was directly applied onto an ischemic injury in the hind limb of mice. The transplanted HUVECs as a sheet retarded tissue necrosis over 14 days in comparison with that of direct injection of the same number of cells. Our results suggest that the developed multifunctional Tetronic-tyramine hydrogels could serve as an ideal substrate to modulate the formation of an endothelial cell layer that could potentially be utilized to treat peripheral arterial disease.

Polydopamine-mediated surface modification of scaffold materials for human neural stem cell 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.

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.

Electroactive electrospun polyaniline/poly[(L-lactide)-co-(ε-caprolactone)] fibers for control of neural cell function.

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