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.

Femtosecond laser induced nano-textured micropatterning to regulate cell functions on implanted biomaterials.

Posterior capsular opacification (PCO) is the most common complication of cataract surgery. PCO is due to the proliferation, migration, and epithelial-to-mesenchymal transition of the residual lens epithelial cells (LECs) within the lens capsule. As surface topography influences cellular response, we investigated the effect of modulating the dimensions of periodic nano-textured patterns on the surface of an intraocular lens material to regulate lens epithelial cell functions such as cell adhesion, migration, orientation, and proliferation. Patterned poly(HEMA) samples were prepared by a femtosecond laser microfabrication, and the behaviors of human B-3 LECs were observed on groove/ridge patterns with widths varying from 5 to 40 µm. In the presence of ridge and groove patterns, the adherent cells elongated along the direction of the patterns, and f-actin of the cells was spread to a lesser extent on the nano-textured groove surfaces. Both single and collective cell migrations were significantly inhibited in the perpendicular direction of the patterns on the nano-textured micro-patterned samples. We also fabricated the patterns on the curved surface of a commercially available intraocular lens for in vivo evaluation. In vivo results showed that a patterned IOL could help suppress the progression of PCO by inhibiting cell migration from the edge to the center of the IOL. Our reports demonstrate that nano- and microscale topographical patterns on a biomaterial surface can regulate cellular behavior when it is implanted into animals.

Biodegradable Magnesium Alloys Promote Angio-Osteogenesis to Enhance Bone Repair.

Biodegradable metallic materials represent a potential step-change technology that may revolutionize the treatment of broken bones. Implants made with biodegradable metals are significantly stronger than their polymer counterparts and fully biodegradable in vivo, removing the need for secondary surgery or long-term complications. Here, it is shown how clinically approved Mg alloy promotes improved bone repair using an integrated state of the art fetal mouse metatarsal assay coupled with in vivo preclinical studies, second harmonic generation, secretome array analysis, perfusion bioreactor, and high-resolution 3D confocal imaging of vasculature within skeletal tissue, to reveal a vascular-mediated pro-osteogenic mechanism controlling enhanced tissue regeneration. The optimized mechanical properties and corrosion rate of the Mg alloy lead to a controlled release of metallic Mg, Ca, and Zn ions at a rate that facilitates both angiogenesis and coupled osteogenesis for better bone healing, without causing adverse effects at the implantation site. The findings from this study support ongoing development and refinement of biodegradable metal systems to act as crucial portal technologies with significant potential to improve many clinical applications.

Tailoring H2O2 generation kinetics with magnesium alloys for efficient disinfection on titanium surface.

A new antibacterial strategy for Ti has been developed without the use of any external antibacterial agents and surface treatments. By combining Mg alloys with Ti, H2O2, which is an oxidizing agent that kills bacteria, was spontaneously generated near the surface of Ti. Importantly, the H2O2 formation kinetics can be precisely controlled by tailoring the degradation rates of Mg alloys connected to Ti. Through microstructural and electrochemical modification of Mg with alloying elements (Ca, Zn), the degradation rates of Mg alloys were controlled, and the H2O2 release kinetics was accelerated when the degradation rate of Mg alloys increased. With the introduction of an in vivo assessment platform comprised of Escherichia coli (E. coli) and transgenic zebrafish embryos, we are able to design optimized antibacterial systems (Ti-Mg and Ti-Mg-3wt% Zn) that can selectively eradicate E. coli while not harming the survival rate, development, and biological functions of zebrafish embryos. We envision that our antibacterial strategy based on utilization of sacrificial Mg alloys could broaden the current palette of antibacterial platforms for metals.

An Ultrastretchable and Self-Healable Nanocomposite Conductor Enabled by Autonomously Percolative Electrical Pathways.

Both self-healable conductors and stretchable conductors have been previously reported. However, it is still difficult to simultaneously achieve high stretchability, high conductivity, and self-healability. Here, we observed an intriguing phenomenon, termed "electrical self-boosting", which enables reconstructing of electrically percolative pathways in an ultrastretchable and self-healable nanocomposite conductor (over 1700% strain). The autonomously reconstructed percolative pathways were directly verified by using microcomputed tomography and in situ scanning electron microscopy. The encapsulated nanocomposite conductor shows exceptional conductivity (average value: 2578 S cm-1; highest value: 3086 S cm-1) at 3500% tensile strain by virtue of efficient strain energy dissipation of the self-healing polymer and self-alignment and rearrangement of silver flakes surrounded by spontaneously formed silver nanoparticles and their self-assembly in the strained self-healing polymer matrix. In addition, the conductor maintains high conductivity and stretchability even after recovered from a complete cut. Besides, a design of double-layered conductor enabled by the self-bonding assembly allowed a conducting interface to be located on the neutral mechanical plane, showing extremely durable operations in a cyclic stretching test. Finally, we successfully demonstrated that electromyogram signals can be monitored by our self-healable interconnects. Such information was transmitted to a prosthetic robot to control various hand motions for robust interactive human-robot interfaces.

Interface Engineering of Fully Metallic Stents Enabling Controllable H2O2 Generation for Antirestenosis.

Despite significant advances in the design of metallic materials for bare metal stents (BMSs), restenosis induced by the accumulation of smooth muscle cells (SMCs) has been a major constraint on improving the clinical efficacy of stent implantation. Here, a new strategy for avoiding this issue by utilizing hydrogen peroxide (H2O2) generated by the galvanic coupling of nitinol (NiTi) stents and biodegradable magnesium-zinc (Mg-Zn) alloys is reported. The amount of H2O2 released is carefully optimized via the biodegradability engineering of the alloys and by controlling the immersion time to selectively inhibit the proliferation and function of SMCs without harming vascular endothelial cells. Based on demonstrations of its unique capabilities, a fully metallic stent with antirestenotic functionality was successfully fabricated by depositing Mg layers onto commercialized NiTi stents. The introduction of surface engineering to yield a patterned Mg coating ensured the maintenance of a stable interface between Mg and NiTi during the process of NiTi stent expansion, showing high feasibility for clinical application. This new concept of an inert metal/degradable metal hybrid system based on galvanic metal coupling, biodegradability engineering, and surface patterning can serve as a novel way to construct functional and stable BMSs for preventing restenosis.

A new corrosion-inhibiting strategy for biodegradable magnesium: reduced nicotinamide adenine dinucleotide (NADH).

Utilization of biodegradable metals in biomedical fields is emerging because it avoids high-risk and uneconomic secondary surgeries for removing implantable devices. Mg and its alloys are considered optimum materials for biodegradable implantable devices because of their high biocompatibility; however, their excessive and uncontrollable biodegradation is a difficult challenge to overcome. Here, we present a novel method of inhibiting Mg biodegradation by utilizing reduced nicotinamide adenine dinucleotide (NADH), an endogenous cofactor present in all living cells. Incorporating NADH significantly increases Mg corrosion resistance by promoting the formation of thick and dense protective layers. The unique mechanism by which NADH enables corrosion inhibition was discovered by combined microscopic and spectroscopic analyses. NADH is initially self-adsorbed onto the surface of Mg oxide layers, preventing Cl- ions from dissolving Mg oxides, and later recruits Ca2+ ions to form stable Ca-P protective layers. Furthermore, stability of NADH as a corrosion inhibitor of Mg under physiological conditions were confirmed using cell tests. Moreover, excellent cell adhesion and viability to Mg treated with NADH shows the feasibility of introduction of NADH to Mg-based implantable system. Our strategy using NADH suggests an interesting new way of delaying the degradation of Mg and demonstrates potential roles for biomolecules in the engineering the biodegradability of metals.

Transgenic zebrafish model for quantification and visualization of tissue toxicity caused by alloying elements in newly developed biodegradable metal.

The cytotoxicity of alloying elements in newly developed biodegradable metals can be assessed through relatively low-cost and rapid in vitro studies using different cell types. However, such approaches have limitations; as such, additional investigations in small mammalian models are required that recapitulate the physiological environment. In this study, we established a zebrafish (Danio rerio) model for cytotoxicity evaluations that combines the physiological aspects of an animal model with the speed and simplicity of a cell-based assay. The model was used to assess the cytotoxicity of five common alloying elements in biodegradable implant materials. Conventional in vitro testing using heart, liver, and endothelial cell lines performed in parallel with zebrafish studies revealed statistically significant differences in toxicity (up to 100-fold), along with distinct changes in the morphology of the heart, liver, and blood vessels that were undetectable in cell cultures. These results indicate that our zebrafish model is a useful alternative to mammalian systems for accurately and rapidly evaluating the in vivo toxicity of newly developed metallic materials.

Effect of spatial arrangement and structure of hierarchically patterned fibrous scaffolds generated by a femtosecond laser on cardiomyoblast behavior.

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.

Erratum: Direct and accurate measurement of size dependent wetting behaviors for sessile water droplets.

This corrects the article DOI: 10.1038/srep18150.

Comprehensive study on the roles of released ions from biodegradable Mg-5 wt% Ca-1 wt% Zn alloy in bone regeneration.

We report here the effect of micro-environmental changes from biodegradable magnesium alloys on the activities of cells - osteoblasts, osteoclasts and macrophages - which play critical roles in each phase of the bone-regeneration process. Despite positive bone formation effects from several in vivo studies, minimal progress has been made in identifying underlying mechanisms through in vitro studies, which are currently concentrated on osteoblastic activities. The observed in vitro and in vivo results indicated that alkaline pH and released magnesium and zinc ions derived from Mg-5 wt% Ca-1 wt% Zn alloy biodegradation promote the progress of bone formation. In contrast, alkaline pH and magnesium ions remarkably suppressed osteoclastic activities and pro-inflammatory cytokine production, closely related to osteolysis and prosthesis failure. Findings from the present study conclude that the degradation of Mg-5 wt% Ca-1 wt% Zn alloys can promote new bone formation by simultaneously affecting the complex combination of variable cellular activities and phases. Copyright © 2016 John Wiley & Sons, Ltd.

Label-free bacterial detection using polydiacetylene liposomes.

Polydiacetylene (PDA) liposomes were prepared to selectively capture target released from bacteria. Specific interplay between released-surfactin and PDA resulted in a conformal change in the structure of PDA, highlighting the potential of indirect interactions between bacteria and PDA in the construction of new label-free bacterial sensors.

Ultrathin Metal Films with Defined Topographical Structures as In Vitro Cell Culture Platforms for Unveiling Vascular Cell Behaviors.

Implanted material surfaces make direct contact with body tissues to work on its own purpose. Therefore, studies of the surface properties of implantable materials that determine cell fate are very important for successful implantation. Although numerous studies have addressed the relationship between cells and material surfaces, nonmetallic surfaces and metallic surfaces likely produce different cellular responses because of their intrinsic differences in surface energy, roughness, and chemical composition. Moreover, given the nontransparent property of metal materials, which hampers the real-time imaging of cellular behavior, a detailed cellular-level analysis at the metal-tissue interface has not been performed. In this study, metal-based cell culture platforms (MCPs) with defined microscale topographical patterns are developed using a combination of photolithography and direct current magnetron sputtering techniques. The MCPs allow to observe vascular cells on metals in real time and identify the selective regulation of human aortic smooth muscle cells and Human umbilical vein endothelial cells (HUVECs) by metallic surface topography. Additionally, atomic force microscopy, contact angles, and energy-dispersive X-ray spectroscopy analyses show that the MCPs exhibit nearly identical chemical properties with their bulk counterparts, demonstrating that MCPs can be utilized as an in vitro cell culture platform system for understanding the cellular behavior on metal substrates.

Long-term clinical study and multiscale analysis of in vivo biodegradation mechanism of Mg alloy.

There has been a tremendous amount of research in the past decade to optimize the mechanical properties and degradation behavior of the biodegradable Mg alloy for orthopedic implant. Despite the feasibility of degrading implant, the lack of fundamental understanding about biocompatibility and underlying bone formation mechanism is currently limiting the use in clinical applications. Herein, we report the result of long-term clinical study and systematic investigation of bone formation mechanism of the biodegradable Mg-5wt%Ca-1wt%Zn alloy implant through simultaneous observation of changes in element composition and crystallinity within degrading interface at hierarchical levels. Controlled degradation of Mg-5wt%Ca-1wt%Zn alloy results in the formation of biomimicking calcification matrix at the degrading interface to initiate the bone formation process. This process facilitates early bone healing and allows the complete replacement of biodegradable Mg implant by the new bone within 1 y of implantation, as demonstrated in 53 cases of successful long-term clinical study.

Creating Hierarchical Topographies on Fibrous Platforms Using Femtosecond Laser Ablation for Directing Myoblasts Behavior.

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

Magnesium ions facilitate integrin alpha 2- and alpha 3-mediated proliferation and enhance alkaline phosphatase expression and activity in hBMSCs.

Magnesium metal and its alloys have been proposed as a novel class of bone implant biomaterials because of their biodegradability and mechanical properties. The purpose of this study was to determine whether magnesium ions, which are released abundantly from alloys, affect proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs). High levels of magnesium ions did not induce cytotoxicity in hBMSCs, but treatment with 2.5-10 mm magnesium ions for 48-72 h significantly increased hBMSC proliferation. The expression of integrins α2 and α3, but not ß1, was upregulated compared with the control and shifted from α3 to α2 in hBMSCs treated with magnesium ions. Knockdown of integrins α2 and/or α3 significantly reduced magnesium-induced proliferation of hBMSCs. Magnesium exposure profoundly enhanced alkaline phosphatase (ALP) gene expression and activity even at a relatively low magnesium concentration (2.5 mm). Exposure to magnesium ions facilitated hBMSC proliferation via integrin α2 and α3 expression and partly promoted differentiation into osteoblasts via the alteration of ALP expression and activity. Accordingly, magnesium could be a useful biomaterial for orthopaedic applications such as bone implant biomaterials for repair and regeneration of bone defects in orthopaedic and dental fields. Copyright © 2014 John Wiley & Sons, Ltd.

Nanoparticle coating on the silane-modified surface of magnesium for local drug delivery and controlled corrosion.

In this study, we proposed a potential method for the preparation of a magnesium-based medical device for local drug delivery and controlled corrosion. A magnesium surface was modified with 3-aminopropyltrimethoxy silane, and the resulting surface was then coated with drug-loaded nanoparticles made of poly (lactic-co-glycolic acid) via electrophoretic deposition. The drug-loaded nanoparticles (i.e., Tr_NP) exhibited a size of 250 ± 67 nm and a negative zeta potential of -20.9 ± 2.75 mV. The drug was released from the nanoparticles in a sustained manner for 21 days, and this did not change after their coating on the silane-modified magnesium. The silane-modified surface suppressed magnesium corrosion. When immersed in phosphate buffered saline at pH 7.4, the average rate of hydrogen gas generation was 0.41-0.45 ml/cm(2)/day, compared to 0.58-0.6 ml/cm(2)/day from a bare magnesium surface. This corrosion profile was not significantly changed after nanoparticle coating under the conditions employed in this work. The in vitro cell test revealed that the drug released from the coating was effective during the whole release period of 21 days, and both the silane-modified surface and carrier nanoparticles herein were not cytotoxic.

Direct and accurate measurement of size dependent wetting behaviors for sessile water droplets.

The size-dependent wettability of sessile water droplets is an important matter in wetting science. Although extensive studies have explored this problem, it has been difficult to obtain empirical data for microscale sessile droplets at a wide range of diameters because of the flaws resulting from evaporation and insufficient imaging resolution. Herein, we present the size-dependent quantitative change of wettability by directly visualizing the three phase interfaces of droplets using a cryogenic-focused ion beam milling and SEM-imaging technique. With the fundamental understanding of the formation pathway, evaporation, freezing, and contact angle hysteresis for sessile droplets, microdroplets with diameters spanning more than three orders of magnitude on various metal substrates were examined. Wetting nature can gradually change from hydrophobic at the hundreds-of-microns scale to super-hydrophobic at the sub-µm scale, and a nonlinear relationship between the cosine of the contact angle and contact line curvature in microscale water droplets was demonstrated. We also showed that the wettability could be further tuned in a size-dependent manner by introducing regular heterogeneities to the substrate.

Magnesium Corrosion Triggered Spontaneous Generation of H2O2 on Oxidized Titanium for Promoting Angiogenesis.

Although the use of reactive oxygen species (ROS) has been extensively studied, current systems employ external stimuli such as light or electrical energy to produce ROS, which limits their practical usage. In this report, biocompatible metals were used to construct a novel electrochemical system that can spontaneously generate H2O2 without any external light or voltage. The corrosion of Mg transfers electrons to Au-decorated oxidized Ti in an energetically favorable process, and the spontaneous generation of H2O2 in an oxygen reduction reaction was revealed to occur at titanium by combined spectroscopic and electrochemical analyses. The controlled release of H2O2 noticeably enhanced in vitro angiogenesis even in the absence of growth factors. Finally, a new titanium implant prototype was developed by Mg incorporation, and its potential for promoting angiogenesis was demonstrated.