Dexter Siu Hong Wong

Magnetically Tuning Tether Mobility of Integrin Ligand Regulates Adhesion, Spreading, and Differentiation of Stem Cells

Cells sense and respond to the surrounding microenvironment through binding of membranous integrin to ligands such as the Arg-Gly-Asp (RGD) peptide. Previous studies show that the RGD tether properties on substrate influence cell adhesion and spreading, but few studies have reported strategies to control the tether mobility of RGD on substrate via a physical and noncontact approach. Herein, we demonstrate a novel strategy to tune the tether mobility of RGD on substrate via magnetic force. We conjugate a monolayer of RGD-bearing magnetic nanoparticles (MNPs) on a glass substrate via the flexible and coiled poly(ethylene glycol) linker of large molecular weight (PEG, average MW: 2000), and this increases the RGD tether mobility, which can be significantly reduced by applying magnetic attraction on MNPs. Our data show that high RGD tether mobility delays the early adhesion and spreading of human mesenchymal stem cells (hMSCs), leading to compromised osteogenic differentiation at later stage. In contrast, hMSCs cultured on substrate with restricted RGD tether mobility, achieved either via a shorter PEG linker (MW: 200) or magnetic force, show significantly better adhesion, spreading, and osteogenic differentiation. The control utilizing RGD-bearing nonmagnetic nanoparticles shows no such enhancing effect of magnetic field on cellular events, further supporting our conjecture of magnetic tuning of RGD tether mobility. We hypothesize that high tether mobility of RGD entails additional time and effort by the cells to fully develop traction force and mechanical feedback, thereby delaying the maturation of FAs and activation of subsequent mechanotransduction signaling. Our staining results of vinculin, a critical component of FAs, and Yes-associated protein (YAP), an important mechanosensitive transcriptional factor, support our hypothesis. We believe that our work not only sheds light on the impact of dynamic presentation of cell adhesive ligands on cellular behaviors, which should be taken into consideration for designing novel biomaterials, but also formulate an effective noncontact strategy that enables further investigation on the mechanobiological mechanisms underlying such cellular responses.

Photocontrolled SiRNA Delivery and Biomarker-Triggered Luminogens of Aggregation-Induced Emission by Up-Conversion NaYF4:Yb3+Tm3+@SiO2 Nanoparticles for Inducing and Monitoring Stem-Cell Differentiation

Controlling the differentiation of stem cells and monitoring cell differentiation has attracted much research interest since the discovery of stem cells. In this regard, a novel near-infrared (NIR) light-activated nanoplatform is obtained by encapsulating the photoactivatable caged compound (DMNPE/siRNA) and combining a MMP13 cleaved imaging peptide-tetrapheny-lethene (TPE) unit conjugated with the mesoporous silica-coated up-conversion nanoparticles (UCNPs) for the remote control of cell differentiation and, simultaneously, for the real-time monitoring of differentiation. Upon NIR light illumination, the photoactivated caged compound is activated, and the siRNA is released from UCNPs, allowing controlled differentiation of stem cells by light. More importantly, MMP13 enzyme triggered by osteogenic differentiation would effectively cleave the TPE probe peptide, thereby allowing the real-time monitoring of differentiation in living stem cells by aggregation-induced emission (AIE).

Remote Control of Multimodal Nanoscale Ligand Oscillations Regulates Stem Cell Adhesion and Differentiation

Cellular adhesion is regulated by the dynamic ligation process of surface receptors, such as integrin, to adhesive motifs, such as Arg-Gly-Asp (RGD). Remote control of adhesive ligand presentation using external stimuli is an appealing strategy for the temporal regulation of cell-implant interactions in vivo and was recently demonstrated using photochemical reaction. However, the limited tissue penetration of light potentially hampers the widespread applications of this method in vivo. Here, we present a strategy for modulating the nanoscale oscillations of an integrin ligand simply and solely by adjusting the frequency of an oscillating magnetic field to regulate the adhesion and differentiation of stem cells. A superparamagnetic iron oxide nanoparticle (SPION) was conjugated with the RGD ligand and anchored to a glass substrate by a long flexible poly(ethylene glycol) linker to allow the oscillatory motion of the ligand to be magnetically tuned. In situ magnetic scanning transmission electron microscopy and atomic force microscopy imaging confirmed the nanoscale motion of the substrate-tethered RGD-grafted SPION. Our findings show that ligand oscillations under a low oscillation frequency (0.1 Hz) of the magnetic field promoted integrin-ligand binding and the formation and maturation of focal adhesions and therefore the substrate adhesion of stem cells, while ligands oscillating under high frequency (2 Hz) inhibited integrin ligation and stem cell adhesion, both in vitro and in vivo. Temporal switching of the multimodal ligand oscillations between low- and high-frequency modes reversibly regulated stem cell adhesion. The ligand oscillations further induced the stem cell differentiation and mechanosensing in the same frequency-dependent manner. Our study demonstrates a noninvasive, penetrative, and tunable approach to regulate cellular responses to biomaterials in vivo. Our work not only provides additional insight into the design considerations of biomaterials to control cellular adhesion in vivo but also offers a platform to elucidate the fundamental understanding of the dynamic integrin-ligand binding that regulates the adhesion, differentiation, and mechanotransduction of stem cells.

Optical µ-Printing of Cellular-Scale Microscaffold Arrays for 3D Cell Culture

Guiding cell culture via engineering extracellular microenvironment has attracted tremendous attention due to its appealing potentials in the repair, maintenance, and development of tissues or even whole organs. However, conventional biofabrication technologies are usually less productive in fabricating microscale three-dimensional (3D) constructs because of the strident requirements in processing precision and complexity. Here we present an optical µ-printing technology to rapidly fabricate 3D microscaffold arrays for 3D cell culture and cell-scaffold interaction studies on a single chip. Arrays of 3D cubic microscaffolds with cubical sizes matching the single-cell size were fabricated to facilitate cell spreading on suspended microbeams so as to expose both apical and basal cell membranes. We further showed that the increasing of the cubical size of the microscaffolds led to enhanced spreading of the seeded human mesenchymal stem cells and activation of mechanosensing signaling, thereby promoting osteogenesis. Moreover, we demonstrated that the spatially selective modification of the surfaces of suspended beams with a bioactive coating (gelatin methacrylate) via an in-situ printing process allowed tailorable cell adhesion and spreading on the 3D microscaffolds.

Remote Manipulation of Ligand Nano-Oscillations Regulates Adhesion and Polarization of Macrophages in Vivo

Macrophages play crucial roles in various immune-related responses, such as host defense, wound healing, disease progression, and tissue regeneration. Macrophages perform distinct and dynamic functions in vivo, depending on their polarization states, such as the pro-inflammatory M1 phenotype and pro-healing M2 phenotype. Remote manipulation of the adhesion of host macrophages to the implants and their subsequent polarization in vivo can be an attractive strategy to control macrophage polarization-specific functions but has rarely been achieved. In this study, we grafted RGD ligand-bearing superparamagnetic iron oxide nanoparticles (SPIONs) to a planar matrix via a long flexible linker. We characterized the nanoscale motion of the RGD-bearing SPIONs grafted to the matrix, in real time by in situ magnetic scanning transmission electron microscopy (STEM) and in situ atomic force microscopy. The magnetic field was applied at various oscillation frequencies to manipulate the frequency-dependent ligand nano-oscillation speeds of the RGD-bearing SPIONs. We demonstrate that a low oscillation frequency of the magnetic field stimulated the adhesion and M2 polarization of macrophages, whereas a high oscillation frequency suppressed the adhesion of macrophages but promoted their M1 polarization, both in vitro and in vivo. Macrophage adhesion was also temporally regulated by switching between the low and high frequencies of the oscillating magnetic field. To the best of our knowledge, this is the first demonstration of the remote manipulation of the adhesion and polarization phenotype of macrophages, both in vitro and in vivo. Our system offers the promising potential to manipulate host immune responses to implanted biomaterials, including inflammation or tissue reparative processes, by regulating macrophage adhesion and polarization.

Magnetic Manipulation of Reversible Nanocaging Controls In Vivo Adhesion and Polarization of Macrophages

Macrophages are key immune cells that perform various physiological functions, such as the maintenance of homeostasis, host defense, disease progression, and tissue regeneration. Macrophages adopt distinctly polarized phenotypes, such as pro-inflammatory M1 phenotype or anti-inflammatory (pro-healing) M2 phenotype, to execute disparate functions. The remotely controlled reversible uncaging of bioactive ligands, such as Arg-Gly-Asp (RGD) peptide, is an appealing approach for temporally regulating the adhesion and resultant polarization of macrophages on implants in vivo. Here, we utilize physical and reversible uncaging of RGD by a magnetic field that allows facile tissue penetration. We first conjugated a RGD-bearing gold nanoparticle (GNP) to the substrate and then a magnetic nanocage (MNC) to the GNP via a flexible linker to form the heterodimeric nanostructure. We magnetically manipulated nanoscale displacement of MNC and thus its proximity to the GNP to reversibly uncage and cage RGD. The uncaging of RGD temporally promoted the adhesion and subsequent M2 polarization of macrophages while inhibiting their M1 polarization both in vitro and in vivo. The RGD uncaging-mediated adhesion and M2 polarization of macrophages involved rho-associated protein kinase signaling. This study demonstrates physical and reversible uncaging of RGD to regulate the adhesion and polarization of host macrophages in vivo. This approach of magnetically regulating the heterodimer conformation for physical and reversible uncaging of RGD offers the promising potential to manipulate inflammatory or tissue-regenerative immune responses to the implants in vivo.

Remote Control of Heterodimeric Magnetic Nanoswitch Regulates the Adhesion and Differentiation of Stem Cells

Remote, noninvasive, and reversible control over the nanoscale presentation of bioactive ligands, such as Arg-Gly-Asp (RGD) peptide, is highly desirable for temporally regulating cellular functions in vivo. Herein, we present a novel strategy for physically uncaging RGD using a magnetic field that allows safe and deep tissue penetration. We developed a heterodimeric nanoswitch consisting of a magnetic nanocage (MNC) coupled to an underlying RGD-coated gold nanoparticle (AuNP) via a long flexible linker. Magnetically controlled movement of MNC relative to AuNP allowed reversible uncaging and caging of RGD that modulate physical accessibility of RGD for integrin binding, thereby regulating stem cell adhesion, both in vitro and in vivo. Reversible RGD uncaging by the magnetic nanoswitch allowed temporal regulation of stem cell adhesion, differentiation, and mechanosensing. This physical and reversible RGD uncaging utilizing heterodimeric magnetic nanoswitch is unprecedented and holds promise in the remote control of cellular behaviors in vivo.

Citrate-based fluorophores in polymeric matrix by easy and green in situ synthesis for full-band UV shielding and emissive transparent display

Developing easy and green strategy to prepare functional materials with outstanding properties based on naturally abundant and environmentally friendly raw materials is highly desirable for sustainable development. Herein, an easy and green strategy was reported to in situ synthesize and disperse citrate-based fluorophores (CFs) in polyvinyl alcohol (PVA) matrix. By a simple heating treatment of the mixture aqueous solution of citric acid/cysteine/PVA, CF–PVA blends were obtained in the form of homogeneous transparent films or coatings. Due to the effective UV absorption of CFs, CF–PVA film and coating exhibited full-band blocking of UV irradiation while still allowing high transmission of visible light. Protection by CF–PVA film and coating effectively reduced UV-induced rhodamine B degradation and cell death. Furthermore, the down-conversion property of CF–PVA coating enables conversion of invisible UV irradiation into visible blue light emission, and we further demonstrated the application of CF–PVA coating for fabricating emissive transparent display. Given the abundant and environmentally friendly raw materials, easy and green preparation, excellent UV-blocking, and converting properties, we believe that the CF–PVA blends are promising for applications in UV shielding, transparent display, and energy harvesting.

Detection of Matrix Metallopeptidase 13 for Monitoring Stem Cell Differentiation and Early Diagnosis of Osteoarthritis by Fluorescent Light-Up Probes with Aggregation-Induced Emission Characteristics

Osteoarthritis (OA) is the leading cause of chronic disability affecting the elderly. There is an acute demand for novel approaches to sensitively and specifically detect OA biomarkers in conjunction with traditional radiographic outcomes to facilitate early diagnosis and allow timely treatment. In this study, a novel strategy is introduced to detect the activity of matrix metallopeptidase 13 (MMP‐13)—a key enzyme responsible for cartilage matrix degradation in OA with a synthesized molecular probe containing a hydrophilic MMP‐13‐sensitive peptide conjugated to an aggregation induced emission fluorogen (AIEgen). The MMP‐13 cleaves the MMP‐13 sensitive peptide and induces aggregation of the hydrophogic AIEgen residues resulting in the activation of a fluorescent signal. The results demonstrate that this probe can detect increasing MMP‐13 activity, which is an important marker of osteogenic differentiation in living and differentiating stem cells. This allows easy and semi‐quantitative assessment of the extent of stem cell differentiation. Furthermore, by administering this probe to diseased joints of rats with induced OA, the real‐time detection of MMP‐13 activity is demonstrated in the osteoarthritic knee joints of living animals. It is believed that the molecular probe is a promising tool for real‐time detection of disease markers with high fluorescence contrast to aid the early diagnosis of OA.

Direct optical patterning of poly(dimethylsiloxane) microstructures for microfluidic chips

In this paper, we present an optical maskless exposure approach for direct patterning of large-area high resolution microfluidic chips using photosensitive poly(dimethylsiloxane) (PDMS) materials. Both positive- and negative-tone photosensitive PDMS (photoPDMS) were successfully patterned into various microfluidic devices with complex geometries by using an optical maskless lithography process. The positive-tone PDMS is used for patterning of largearea chips, while the negative-tone PDMS is demonstrated to fabricate high-resolution microstructures and on-chip devices. With the seamless pattern-stitching technique, a large-area microfluidic chip with size of 5.5 × 2.8 cm2 with complex three-dimensional (3D) staggered herringbone mixers (SHMs) for micro-flow gradient generation has been directly fabricated within 125 minutes by using the positive-tone PDMS. A small microfluidic chip with feature size as small as 5 μm is demonstrated by using the negative-tone PDMS. The experimental results reveal that the optical maskless lithography technology enables to rapidly pattern high-resolution microstructures and is very promising for development of lab-on-a-chip devices.