Sequences of primers and probes used for Real-Time PCR. Sequences related to gene type X collagen and MMP13 are proprietary to Applied Biosystems Inc. and not disclosed.

<p>Sequences of primers and probes used for Real-Time PCR. Sequences related to gene type X collagen and MMP13 are proprietary to Applied Biosystems Inc. and not disclosed.</p

The evaluation of the hypertrophic calcification of the hydrogels.

<p>Calcium content of the hMSC-laden hydrogels normalized to wet weight (w.t.) and dry weight (d.w) (A); and Von Kossa staining of the histological sections of the hydrogels after 28 days of culture, bar  = 500 µm, *p<0.05 vs. MMP group (n = 4).</p

The gene expression of type II collagen, aggrecan, type X collagen and MMP13 in fold changes on day 7, 14 or 28 of the culture normalized to pre-differentiated cells.

<p>*p<0.05 vs. MMP group (n = 4).</p

The analysis of the cartilaginous matrix contents of the hMSC-seeded hydrogels.

<p>GAG and collagen content (normalized to the DNA content) of hMSC-laden HA hydrogels (A) and immunohistochemistry staining of the hydrogel constructs against chondroitin sulfate, type II and I collagen after 28 days of culture (B), Bar  = 50 µm, *p<0.05 vs. MMP group (n = 4).</p

The cell viability staining on day 1 (A) and 14 (B) of the culture.

<p>Bar  = 100 µm.</p

Dual Molecular Recognition Leading to a Protein–Polymer Conjugate and Further Self-Assembly

Supramolecular conjugation between native protein concanavalin A (ConA) and synthetic polymer PEG (polyethylene glycol) was achieved by dual molecular recognition interactions via a linker, βCD-Man, of which β-cyclodextrin (βCD) and α-mannopyranoside (Man) recognized the adamantane (Ada) end of PEG and lectin ConA orthogonally. Further self-assembly of the resultant supra-conjugates of ConA-PEG was induced by the addition of αCD, which was selectively threaded by PEG chains, leading to nanoparticles in dilute solution or hydrogel at a higher concentration. The moduli of the obtained hydrogel were three magnitudes higher than those of the control sample without ConA, showing the dramatic cross-linking effect of ConA achieved by its rather weak interaction with α-d-mannopyranoside

Multivalent Host–Guest Hydrogels as Fatigue-Resistant 3D Matrix for Excessive Mechanical Stimulation of Encapsulated Cells

Fatigue resistance of hydrogels is critical to their applications in load-bearing sites of soft tissues that are usually subjected to continuous loadings, such as joint cartilage. However, hydrogels usually swell under physiological conditions and exhibit inevitable fatigue during excessive mechanical loadings. Here we show that hydrogels cross-linked by multivalent host–guest interactions can effectively dissipate a large fraction of the loading energy (>50%) under excessive compressions (over 80% strain, 1000 cycles) despite their high water contents (95%) under physiological conditions. No fatigue is observed in such highly swollen hydrogels during continuous cyclic compressions. We demonstrate that such hydrogels can be used as 3D cell carriers for excessive mechanical stimulation of the encapsulated stem cells, making them promising soft biomaterials for tissue engineering

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 <i>in vivo</i> and was recently demonstrated using photochemical reaction. However, the limited tissue penetration of light potentially hampers the widespread applications of this method <i>in vivo</i>. 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. <i>In situ</i> 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 <i>in vitro</i> and <i>in vivo</i>. 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 <i>in vivo</i>. Our work not only provides additional insight into the design considerations of biomaterials to control cellular adhesion <i>in vivo</i> 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

A Gold@Polydopamine Core–Shell Nanoprobe for Long-Term Intracellular Detection of MicroRNAs in Differentiating Stem Cells

The capability of monitoring the differentiation process in living stem cells is crucial to the understanding of stem cell biology and the practical application of stem-cell-based therapies, yet conventional methods for the analysis of biomarkers related to differentiation require a large number of cells as well as cell lysis. Such requirements lead to the unavoidable loss of cell sources and preclude real-time monitoring of cellular events. In this work, we report the detection of microRNAs (miRNAs) in living human mesenchymal stem cells (hMSCs) by using polydopamine-coated gold nanoparticles (Au@PDA NPs). The PDA shell facilitates the immobilization of fluorescently labeled hairpin DNA strands (hpDNAs) that can recognize specific miRNA targets. The gold core and PDA shell quench the fluorescence of the immobilized hpDNAs, and subsequent binding of the hpDNAs to the target miRNAs leads to their dissociation from Au@PDA NPs and the recovery of fluorescence signals. Remarkably, these Au@PDA–hpDNA nanoprobes can naturally enter stem cells, which are known for their poor transfection efficiency, without the aid of transfection agents. Upon cellular uptake of these nanoprobes, we observe intense and time-dependent fluorescence responses from two important osteogenic marker miRNAs, namely, miR-29b and miR-31, only in hMSCs undergoing osteogenic differentiation and living primary osteoblasts but not in undifferentiated hMSCs and 3T3 fibroblasts. Strikingly, our nanoprobes can afford long-term tracking of miRNAs (5 days) in the differentiating hMSCs without the need of continuously replenishing cell culture medium with fresh nanoprobes. Our results demonstrate the capability of our Au@PDA–hpDNA nanoprobes for monitoring the differentiation status of hMSCs (i.e., differentiating versus undifferentiated) via the detection of specific miRNAs in living stem cells. Our nanoprobes show great promise in the investigation of the long-term dynamics of stem cell differentiation, identification and isolation of specific cell types, and high-throughput drug screening