Bioelectric control of locomotor gaits in the walking ciliate Euplotes.

Diverse animal species exhibit highly stereotyped behavioral actions and locomotor sequences as they explore their natural environments. In many such cases, the neural basis of behavior is well established, where dedicated neural circuitry contributes to the initiation and regulation of certain response sequences. At the microscopic scale, single-celled eukaryotes (protists) also exhibit remarkably complex behaviors and yet are completely devoid of nervous systems. Here, to address the question of how single cells control behavior, we study locomotor patterning in the exemplary hypotrich ciliate Euplotes, a highly polarized cell, which actuates a large number of leg-like appendages called cirri (each a bundle of ∼25-50 cilia) to swim in fluids or walk on surfaces. As it navigates its surroundings, a walking Euplotes cell is routinely observed to perform side-stepping reactions, one of the most sophisticated maneuvers ever observed in a single-celled organism. These are spontaneous and stereotyped reorientation events involving a transient and fast backward motion followed by a turn. Combining high-speed imaging with simultaneous time-resolved electrophysiological recordings, we show that this complex coordinated motion sequence is tightly regulated by rapid membrane depolarization events, which orchestrate the activity of different cirri on the cell. Using machine learning and computer vision methods, we map detailed measurements of cirri dynamics to the cell's membrane bioelectrical activity, revealing a differential response in the front and back cirri. We integrate these measurements with a minimal model to understand how Euplotes-a unicellular organism-manipulates its membrane potential to achieve real-time control over its motor apparatus.

Regulated Behavior in Living Cells with Highly Aligned Configurations on Nanowrinkled Graphene Oxide Substrates: Deep Learning Based on Interplay of Cellular Contact Guidance.

Micro-/nanotopographical cues have emerged as a practical and promising strategy for controlling cell fate and reprogramming, which play a key role as biophysical regulators in diverse cellular processes and behaviors. Extracellular biophysical factors can trigger intracellular physiological signaling via mechanotransduction and promote cellular responses such as cell adhesion, migration, proliferation, gene/protein expression, and differentiation. Here, we engineered a highly ordered nanowrinkled graphene oxide (GO) surface via the mechanical deformation of an ultrathin GO film on an elastomeric substrate to observe specific cellular responses based on surface-mediated topographical cues. The ultrathin GO film on the uniaxially prestrained elastomeric substrate through self-assembly and subsequent compressive force produced GO nanowrinkles with periodic amplitude. To examine the acute cellular behaviors on the GO-based cell interface with nanostructured arrays of wrinkles, we cultured L929 fibroblasts and HT22 hippocampal neuronal cells. As a result, our developed cell-culture substrate obviously provided a directional guidance effect. In addition, based on the observed results, we adapted a deep learning (DL)-based data processing technique to precisely interpret the cell behaviors on the nanowrinkled GO surfaces. According to the learning/transfer learning protocol of the DL network, we detected cell boundaries, elongation, and orientation and quantitatively evaluated cell velocity, traveling distance, displacement, and orientation. The presented experimental results have intriguing implications such that the nanotopographical microenvironment could engineer the living cells' morphological polarization to assemble them into useful tissue chips consisting of multiple cell types.

Novel HIF-1α Inhibitor AMSP-30m Mitigates the Pathogenic Cellular Behaviors of Hypoxia-Stimulated Fibroblast-Like Synoviocytes and Alleviates Collagen-Induced Arthritis in Rats via Inhibiting Sonic Hedgehog Pathway.

Synovial hypoxia-inducible factor 1α (HIF-1α) is a prospective therapeutic target for rheumatoid arthritis (RA). AMSP-30 m, a novel HIF-1α inhibitor, was reported to have notable anti-arthritic effects in rats with adjuvant-induced arthritis. However, its roles in inhibiting the pathogenic behaviors of fibroblast-like synoviocytes (FLS) and the involved mechanisms remain unknown. Here, AMSP-30 m inhibited proliferation and induced apoptosis in hypoxia-induced RA FLS (MH7A cell line), as evidenced by decreased cell viability, reduced Ki67-positive cells, G0/G1 phase arrest, lowered C-myc and Cyclin D1 protein levels, emergence of apoptotic nuclear fragmentation, raised apoptosis rates, and activation of caspase 3. Furthermore, AMSP-30 m prevented hypoxia-induced increases in pro-inflammatory factor production, MMP-2 activity, migration index, migrated/invasive cells, and actin cytoskeletal rearrangement. In vivo, AMSP-30 m alleviated the severity of rat collagen-induced arthritis (CIA). Mechanically, AMSP-30 m reduced HIF-1α expression and blocked sonic hedgehog (Shh) pathway activation in hypoxia-induced MH7A cells and CIA rat synovium, as shown by declines in pathway-related proteins (Shh, Smo, and Gli-1). Particularly, the combination of Shh pathway inhibitor cyclopamine enhanced AMSP-30 m's inhibitory effects on the pathogenic behaviors of hypoxia-stimulated MH7A cells, whereas the combination of Shh pathway activator SAG canceled AMSP-30 m's therapeutic effects in vitro and in CIA rats, implying a close involvement of Shh pathway inhibition in its anti-arthritic effects. We likewise confirmed AMSP-30 m's anti-proliferative role in hypoxia-induced primary CIA FLS. Totally, AMSP-30 m suppressed hypoxia-induced proliferation, inflammation, migration, and invasion of MH7A cells and ameliorated the severity of rat CIA via inhibiting Shh signaling.

Logic Nanodevice-Mediated Receptor Assembly for Nongenetic Regulation of Cell Behavior in Tumor-like Microenvironment.

The reprogramming of cell signaling and behavior through the artificial control of cell surface receptor oligomerization shows great promise in biomedical research and cell-based therapy. However, it remains challenging to achieve combinatorial recognition in a complicated environment and logical regulation of receptors for desirable cellular behavior. Herein, we develop a logic-gated DNA nanodevice with responsiveness to multiple environmental inputs for logically controlled assembly of heterogeneous receptors to modulate signaling. The "AND" gate nanodevice uses an i-motif and an ATP-binding aptamer as environmental cue-responsive units, which can successfully implement a logic operation to manipulate receptors on the cell surface. In the presence of both protons and ATP, the DNA nanodevice is activated to selectively assemble MET and CD71, which modulate the HGF/MET signaling, resulting in cytoskeletal reorganization to inhibit cancer cell motility in a tumor-like microenvironment. Our strategy would be highly promising for precision therapeutics, including controlled drug release and cancer treatment.

Periodontal Ligament-Mimetic Fibrous Scaffolds Regulate YAP-Associated Fibroblast Behaviors and Promote Regeneration of Periodontal Defect in Relation to the Scaffold Topography.

Although multiple regenerative strategies are being developed for periodontal reconstruction, guided periodontal ligament (PDL) regeneration is difficult because of its cellular and fibrous complexities. Here, we manufactured four different types of PDL-mimic fibrous scaffolds on a desired single mat. These scaffolds exhibited a structure of PDL matrix and human PDL fibroblasts (PDLFs) cultured on the scaffolds resembling morphological phenotypes present in native PDLF. The scaffold-seeded PDLF exerted proliferative, osteoblastic, and osteoclastogenic potentials depending on the fiber topographical cues. Fiber surface-regulated behaviors of PDLF were correlated with the expression patterns of yes-associated protein (YAP), CD105, periostin, osteopontin, and vinculin. Transfection with si-RNA confirmed that YAP acted as the master mechanosensing regulator. Of the as-spun scaffolds, aligned or grid-patterned microscale scaffold regulated the YAP-associated behavior of PDLF more effectively than nanomicroscale or random-oriented microscale scaffold. Implantation with hydrogel complex conjugated with microscale-patterned or grid-patterned scaffold, but not other types of scaffolds, recovered the defected PDL with native PDL-mimic cellularization and fiber structure in the reformed PDL. Our results demonstrate that PDL-biomimetic scaffolds regulate topography-related and YAP-mediated behaviors of PDLF in relation to their topographies. Overall, this study may support a clinical approach of the fiber-hydrogel complex in guided PDL regenerative engineering.

QCM sensor provides insight into the role of pivotal ions in cellular regulatory volume decrease.

All vertebrate cells generally self-regulate for sustaining homeostasis and cell functions. As a major regulatory mechanism, regulatory volume decrease (RVD) occurs in hypotonicity-induced cell swelling, and then shrinking by the efflux of intracellular osmolytes and water, in which the ions K+, Cl-, and Ca2+ play a key role in the RVD process. We observed that these pivotal ions could result in novel RVD behaviors under repeatedly hypotonic stimulation. However, there is a lack of valid means for assessing the effect of pivotal ions on RVD. In this work, we proposed an effective measurement process based on a quartz crystal microbalance (QCM) combined with cell function of RVD for revealing acute variations in cell volume regulation induced by the pivotal ions. A QCM sensor was implemented by adhering MCF-7 cells to a poly-l-lysine-modified gold chip and cyclic stimulation with hypotonic NaCl medium, in which a frequency shift (Δf) showed the superior feasibility of the technique in exhibiting RVD behaviors. With the increase in the number of cycles, the RVD values decreased progressively under three stimulation cycles with hypotonic NaCl alone. Compared with the first cycle, the RVD level in the second and third cycles declined by 60.7±1.7% and 82.1±1.6% (n=3), respectively; conversely, it recovered in NaCl-KCl solution, but was significantly enhanced by 52.2±0.8% in NaCl-CaCl2 solution. Moreover, the inhibition of chloride channels to block Cl- efflux also decreased the RVD level by 56.2±3.0%. The results indicate that these ions (K+, Cl-, Ca2+) are all able to affect the function of RVD, among which intracellular Cl- depletion reduced RVD during measurement, but which recovered with K+ supplement, and Ca2+ enhanced RVD due to activation of ion channels. Therefore, this work provides a comprehensive assessment of cellular behavior and offers an innovative method for gaining insight into cellular functions and mechanisms. A novel strategy was conducted by integrating a quartz crystal microbalance (QCM) with the function of cell volume regulation for analyzing the role of the pivotal ions ( K+, Cl-, Ca2+) in NaCl media on the behaviors of regulatory cell volume decrease (RVD).

Spatially Reprogramed Receptor Organization to Switch Cell Behavior Using a DNA Origami-Templated Aptamer Nanoarray.

Receptor oligomerization is a highly complex molecular process that modulates divergent cell signaling. However, there is a lack of molecular tools for systematically interrogating how receptor oligomerization governs the signaling response. Here, we developed a DNA origami-templated aptamer nanoarray (DOTA) that enables precise programming of the oligomerization of receptor tyrosine kinases (RTK) with defined valency, distribution, and stoichiometry at the ligand-receptor interface. The DOTA allows for advanced receptor manipulations by arraying either monomeric aptamer ligands (mALs) that oligamerize receptor monomers to elicit artificial signaling or dimeric aptamer ligands (dALs) that preorganize the receptor dimer to recapitulate natural activation. We demonstrated that the multivalency and nanoscale spacing of receptor oligomerization coordinately influence the activation level of receptor tyrosine kinase signaling. Furthermore, we illustrated that DOTA-modulated receptor oligomerization could function as a signaling switch to promote the transition from epithelia to mesenchymal-like cells, demonstrating robust control over cellular behaviors. Together, we present a versatile all-in-one DNA nanoplatform for the systematical investigation and regulation of receptor-mediated cellular response.

Fabrication of Concave Microwells and Their Applications in Micro-Tissue Engineering: A Review.

At present, there is an increasing need to mimic the in vivo micro-environment in the culture of cells and tissues in micro-tissue engineering. Concave microwells are becoming increasingly popular since they can provide a micro-environment that is closer to the in vivo environment compared to traditional microwells, which can facilitate the culture of cells and tissues. Here, we will summarize the fabrication methods of concave microwells, as well as their applications in micro-tissue engineering. The fabrication methods of concave microwells include traditional methods, such as lithography and etching, thermal reflow of photoresist, laser ablation, precision-computerized numerical control (CNC) milling, and emerging technologies, such as surface tension methods, the deformation of soft membranes, 3D printing, the molding of microbeads, air bubbles, and frozen droplets. The fabrication of concave microwells is transferring from professional microfabrication labs to common biochemical labs to facilitate their applications and provide convenience for users. Concave microwells have mostly been used in organ-on-a-chip models, including the formation and culture of 3D cell aggregates (spheroids, organoids, and embryoids). Researchers have also used microwells to study the influence of substrate topology on cellular behaviors. We will briefly review their applications in different aspects of micro-tissue engineering and discuss the further applications of concave microwells. We believe that building multiorgan-on-a-chip by 3D cell aggregates of different cell lines will be a popular application of concave microwells, while integrating physiologically relevant molecular analyses with the 3D culture platform will be another popular application in the near future. Furthermore, 3D cell aggregates from these biosystems will find more applications in drug screening and xenogeneic implantation.

A unicellular walker controlled by a microtubule-based finite-state machine.

Cells are complex biochemical systems whose behaviors emerge from interactions among myriad molecular components. Computation is often invoked as a general framework for navigating this cellular complexity. However, it is unclear how cells might embody computational processes such that the theories of computation, including finite-state machine models, could be productively applied. Here, we demonstrate finite-state-machine-like processing embodied in cells using the walking behavior of Euplotes eurystomus, a ciliate that walks across surfaces using fourteen motile appendages (cirri). We found that cellular walking entails regulated transitions among a discrete set of gait states. The set of observed transitions decomposes into a small group of high-probability, temporally irreversible transitions and a large group of low-probability, time-symmetric transitions, thus revealing stereotypy in the sequential patterns of state transitions. Simulations and experiments suggest that the sequential logic of the gait is functionally important. Taken together, these findings implicate a finite-state-machine-like process. Cirri are connected by microtubule bundles (fibers), and we found that the dynamics of cirri involved in different state transitions are associated with the structure of the fiber system. Perturbative experiments revealed that the fibers mediate gait coordination, suggesting a mechanical basis of gait control.

Approach to the Fatigue and Cellular Behavior of Superficially Modified Porous Titanium Dental Implants.

In this work, the fatigue and cellular performance of novel superficially treated porous titanium dental implants made up using conventional powder metallurgy and space-holder techniques (30 vol.% and 50 vol.%, both with a spacer size range of 100-200 µm) are evaluated. Before the sintering stage, a specific stage of CNC milling of the screw thread of the implant is used. After the consolidation processing, different surface modifications are performed: chemical etching and bioactive coatings (BG 45S5 and BG 1393). The results are discussed in terms of the effect of the porosity, as well as the surface roughness, chemical composition, and adherence of the coatings on the fatigue resistance and the osteoblast cells' behavior for the proposed implants. Macro-pores are preferential sites of the nucleation of cracks and bone cell adhesion, and they increase the cellular activity of the implants, but decrease the fatigue life. In conclusion, SH 30 vol.% dental implant chemical etching presents the best bio-functional (in vitro osseointegration) and bio-mechanical (stiffness, yield strength and fatigue life) balance, which could ensure the required characteristics of cortical bone tissue.

Influence of Femtosecond Laser Modification on Biomechanical and Biofunctional Behavior of Porous Titanium Substrates.

Bone resorption and inadequate osseointegration are considered the main problems of titanium implants. In this investigation, the texture and surface roughness of porous titanium samples obtained by the space holder technique were modified with a femtosecond Yb-doped fiber laser. Different percentages of porosity (30, 40, 50, and 60 vol.%) and particle range size (100-200 and 355-500 µm) were compared with fully-dense samples obtained by conventional powder metallurgy. After femtosecond laser treatment the formation of a rough surface with micro-columns and micro-holes occurred for all the studied substrates. The surface was covered by ripples over the micro-metric structures. This work evaluates both the influence of the macro-pores inherent to the spacer particles, as well as the micro-columns and the texture generated with the laser, on the wettability of the surface, the cell behavior (adhesion and proliferation of osteoblasts), micro-hardness (instrumented micro-indentation test, P-h curves) and scratch resistance. The titanium sample with 30 vol.% and a pore range size of 100-200 µm was the best candidate for the replacement of small damaged cortical bone tissues, based on its better biomechanical (stiffness and yield strength) and biofunctional balance (bone in-growth and in vitro osseointegration).

The effect of heat stress on the cellular behavior, intracellular signaling profile of porcine growth hormone (pGH) in swine testicular cells.

At present, heat stress caused by the thermal environment is the main factor that endangers the reproductive function of animals. Growth hormone (GH) is a polypeptide hormone, the biological function of reproductive organs has been reported, and it has many important physiological functions in the body. However, so far, the behavior and signal transduction of GH in testicular cells under heat stress are still unclear. To this end, in the current work, we use a swine testicular cell line (ST) as an in vitro model to explore the cell behavior and intracellular signaling profile of porcine growth hormone (pGH) under heat stress; the results showed that when cells were under heat stress, pGH and GHR were basically not internalized, and a large number of them accumulated on the cell membrane. In addition, we also studied the effect of pGH on the JAK2-STATs signaling pathway and IGF-1 expression under heat stress, we found that the ability of pGH to activate the JAK-STATs signaling pathway and IGF-1 under heat stress was greatly reduced (p < 0.05). In conclusion, our research shows that when cells undergo heat stress, the internalization of pGH and GHR were inhibited, and the activation of the JAK2-STATs signaling pathway and IGF-1 expression were reduced; this lays a solid foundation for further research on the effect of pGH on swine testicular tissue under thermal environment.

Conjugated Polymer-Functionalized Stretchable Supramolecular Hydrogels to Monitor and Control Cellular Behavior.

Natural extracellular matrix is formed by the assembly of small molecules and macromolecules into a hydrogel-like network that can mechanically support cells and involve in cellular processes. Here, we developed a fluorescent supramolecular hydrogel based on a conjugated oligomer OFBTCO2Na, which facilitated noncovalent assembly through hydrophobic interactions and hydrogen bonds in a molecular scale. The generated dense three-dimensional network endows the supramolecular hydrogel with stretchability and stability. Furthermore, fluorescent OFBTCO2Na in hydrogel acted as a donor, which can excite the acceptor dyes on cells encapsulated in hydrogel via the Förster resonance energy transfer (FRET) mechanism. Investigating the fluorescence signal responsiveness of hydrogel to dynamic mechanical stretching well reflected that enhanced stretching dictated the extent of connection between the cell and matrix, which enables effective FRET at a molecular level and allow spatiotemporally monitoring cell-matrix interactions at the three-dimensional network. Importantly, cells can sense stretch forces by their connection with a hydrogel matrix. The dynamic cell-matrix interaction can be conveniently employed to formulate cell morphology. Therefore, the fluorescent supramolecular hydrogel offers a suitable culture platform not only to investigate cell interactions on interfaces but also to regulate cell behavior at interfaces.

Upregulation of MicroRNA-937 Predicts a Poor Prognosis and Promotes Hepatocellular Carcinoma Cell Proliferation, Migration, and Invasion.

Hepatocellular carcinoma (HCC) has a high dead rate partly due to late diagnosis. This study aimed to investigate the prognostic value of miR-937 in HCC and its role in the HCC progression. HCC tissue and adjacent non-cancerous tissues (NCT) (n = 125) were detected about the expression level of miR-937 via real-time quantitative PCR. The relationship between miR-937 expression and each important clinical characteristic was evaluated. And the prognostic significance of miR-937 was assessed by Kaplan-Meier curve and Cox regression analysis. CCK-8 and Transwell assays were conducted to observe the effects of miR-937 on HCC cell proliferation, migration, and invasion. The miR-937 expression was upregulated in HCC tissues, as well as in HCC cell lines. The upregulation of miR-937 showed a significant association with lymph node metastasis and TNM stage. Upregulation of miR-937 predicted poor prognosis of HCC patients. Overexpression of miR-937 promoted HCC cell ability of proliferation, migration, and invasiveness, while knockdown of miR-937 inhibited these cellular behaviors. miR-937 expression was upregulated in HCC and may serve as a promising prognostic factor and treated target for HCC patients. miR-937 might exert a promoter role in HCC through accelerated tumor cell proliferation, migration, and invasion.

Poly(amino ester)-Based Polymers for Gene and Drug Delivery Systems and Further Application toward Cell Culture System.

Various synthetic polymers based on poly(amino ester) (PAE) are suggested as candidates for gene and drug delivery owing to their pH-responsiveness, which contributes to efficient delivery performance. PAE-based pH-responsive polymers are more biodegradable and hydrophilic than other types of pH-responsive polymers. The functionality of PAE-based polymers can be reinforced by using different chemical modifications to improve the efficiency of gene and drug delivery. Additionally, PAE-based polymers are used in many ways in the biomedical field, such as in transdermal delivery and stem cell culture systems. Here, the recent novel PAE-based polymers designed for gene and drug delivery systems along with their further applications toward adult stem cell culture systems are reviewed. The synthetic tactics are contemplated and pros and cons of each type of polymer are analyzed, and detailed examples of the different types are analyzed.

SEM and TEM for identification of capsular fibrosis and cellular behavior around breast implants - a descriptive analysis.

BACKGROUND: Capsular fibrosis (CF) is the most common long-term complication in implant-based breast augmentation. It is well accepted that the foreign body response (FBR) instigates the development of fibrotic disease. Our study aims to compare murine and human samples of CF and describe the cellular and extracellular matrix (ECM) composition using scanning and transmission electron microscopy (SEM and TEM). RESULTS: Miniature microtextured silicone breast implants were implanted in mice and subsequently harvested at days 15, 30, and 90 post-operation. Isolated human capsules with the most aggravated form of CF (Baker IV) were harvested post-operation. Both were analyzed with SEM and TEM to assess cellular infiltration and ECM structure. An architectural shift of collagen fiber arrangement from unidirectional to multidirectional was observed at day 90 when compared to days 15 and 30. Fibrosis was observed with an increase of histiocytic infiltration. Moreover, bacterial accumulation was seen around silicone fragments. These findings were common in both murine and human capsules. CONCLUSIONS: This murine model accurately recapitulates CF found in humans and can be utilized for future research on cellular invasion in capsular fibrosis. This descriptive study helps to gain a better understanding of cellular mechanisms involved in the FBR. Increases of ECM and cellularity were observed over time with SEM and TEM analysis.

The Effects of Crosslinking on the Rheology and Cellular Behavior of Polymer-Based 3D-Multilayered Scaffolds for Restoring Articular Cartilage.

Polymer-based tri-layered (bone, intermediate and top layers) scaffolds used for the restoration of articular cartilage were prepared and characterized in this study to emulate the concentration gradient of cartilage. The scaffolds were physically or chemically crosslinked. In order to obtain adequate scaffolds for the intended application, the impact of the type of calcium phosphate used in the bone layer, the polymer used in the intermediate layer and the interlayer crosslinking process were analyzed. The correlation among SEM micrographs, physical-chemical characterization, swelling behavior, rheological measurements and cell studies were examined. Storage moduli at 1 Hz were 0.3-1.7 kPa for physically crosslinked scaffolds, and 4-5 kPa (EDC/NHS system) and 15-20 kPa (glutaraldehyde) for chemically crosslinked scaffolds. Intrinsic viscoelasticity and poroelasticity were considered in discussing the physical mechanism dominating in different time/frequency scales. Cell evaluation showed that all samples are available as alternatives to repair and/or substitute cartilage in articular osteoarthritis.

Customized additive manufacturing of porous Ti6Al4V scaffold with micro-topological structures to regulate cell behavior in bone tissue engineering.

Scaffold micro-topological structure plays an important role in the regulation of cell behavior in bone tissue engineering. This paper investigated the effect of 3D printing parameters on the scaffold micro-topological structure and its subsequent cell behaviors. By setting of different 3D printing parameters, i.e., the 3D printing laser power, the scanning interval and the thickness of sliced layers, the highest resolution up to 20 µm can be precisely fabricated. Scaffolds' characterization results indicated that the laser power affected the forming quality of melt tracks, the scanning interval distance determined the size of regularly arranged pores, and the thickness of sliced layers affected the morphological and structural characteristics. By regulating of these printing parameters, customized porous Ti6Al4V scaffold with varied hierarchical micro-topological structure can be obtained. In vitro cell culturing results showed that the regular porous micro-topological structure of scaffolds with the aperture close to cell size was more suitable for cell proliferation and adhesion. The overall distribution of cells on regular porous scaffolds was similar to the orderly arrangement of cultivated crops in the field. The findings suggested that customization of the scaffold provided an effective way to regulate cellular behavior and biological properties.

Low-intensity pulsed ultrasound promotes the formation of periodontal ligament stem cell sheets and ectopic periodontal tissue regeneration.

Human periodontal ligament stem cells (hPDLSCs) sheets play an important role in periodontal tissue engineering. Low-intensity pulsed ultrasound (LIPUS) has been reported as an effective stimulus to regulate cell biological behavior. The present study aims to explore the potential of LIPUS to promote the formation and function of hPDLSC sheets (hPDLSCSs). Hematoxylin-eosin (H&E) staining, western blot, real-time PCR, alkaline phosphatase (ALP), and alizarin red staining were used to evaluate the formation and osteogenic effect of LIPUS on hPDLSCSs in vitro. Hydroxyapatite with or without hPDLSCSs was transplanted in the subcutaneous pockets on the back of nude mice and histological analysis was performed. H&E staining showed increased synthesis of extracellular matrix (ECM) and real-time PCR detected a significant increase in ECM-related genes after LIPUS treatment. In addition, LIPUS could promote the expression of osteogenic differentiation-related genes and proteins. ALP and alizarin red staining also found LIPUS enhanced the osteogenesis of hPDLSCSs. After transplantation in vivo, more dense collagen fibers similar to periodontal ligament were regenerated. Collectively, these results indicate that LIPUS not only promotes the formation and osteogenic differentiation of hPDLSCSs but also is a potential treatment strategy for periodontal tissue engineering.

Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6ß4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.