Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the critical components accelerating angiogenesis and tissue regeneration by promoting the migration of dermal fibroblasts and endothelial cells associated with matrix formation and remodeling in wound healing process. However, clinical applications of bFGF are substantially limited by its unstable nature due to rapid decomposition under physiological microenvironment. RESULTS: In this study, we present the bFGF-loaded human serum albumin nanoparticles (HSA-bFGF NPs) as a means of enhanced stability and sustained release platform during tissue regeneration. Spherical shape of the HSA-bFGF NPs with uniform size distribution (polydispersity index < 0.2) is obtained via a simple desolvation and crosslinking process. The HSA-bFGF NPs securely load and release the intact soluble bFGF proteins, thereby significantly enhancing the proliferation and migration activity of human dermal fibroblasts. Myofibroblast-related genes and proteins were also significantly down-regulated, indicating decrease in risk of scar formation. Furthermore, wound healing is accelerated while achieving a highly organized extracellular matrix and enhanced angiogenesis in vivo. CONCLUSION: Consequently, the HSA-bFGF NPs are suggested not only as a delivery vehicle but also as a protein stabilizer for effective wound healing and tissue regeneration.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity.

Unjamming and cell shape in the asthmatic airway epithelium.

From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems-both inert and living-have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

Effects of minimal exposures to atmospheric pressure plasma on the activity of Salmonella Typhimurium: Deactivation of bacterial motility and suppression of host-cell invasion.

Atmospheric pressure plasma (APP) has been shown effective in sterilization by reducing the number of viable microbes during surface cleaning, food processing, or human tissue treatment. For safe conduct, the majority of previous research focused on complete abolition of microbes, which may require severe treatments. Our aim is to investigate the minimal treatment conditions necessary for effective inactivation of bacteria in such a manner that the APP treated bacteria would not be able to harm the host cells. For this, we ought to identify the objective criteria to make the bacteria dysfunctional. We choose the motile properties and the host-cell invasion capability as two measures to quantify the pathogenic state of bacteria. In this paper, we investigated how the APP treatment in a minimal dosage affects the activity of Salmonella Typhimurium. At 100 W and 15 kHz for 20 s, the APP treatment effectively suppressed active "run and tumble" type motility and induced formation of abnormally long structures. With 20 s exposure, the bacterial cells failed to cause pyroptosis in the host cells with >90% survival after 12 h of co-incubation. Our results suggest novel measures to evaluate the functional pathogenic state for identifying safe APP treatment conditions.

Propulsion and navigation within the advancing monolayer sheet.

As a wound heals, or a body plan forms, or a tumour invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge, or paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space.

Primary Cilium by Polyinosinic:Polycytidylic Acid Regulates the Regenerative Migration of Beas-2B Bronchial Epithelial Cells.

The airway epithelium is equipped with the ability to resist respiratory disease development and airway damage, including the migration of airway epithelial cells and the activation of TLR3, which recognizes double-stranded (ds) RNA. Primary cilia on airway epithelial cells are involved in the cell cycle and cell differentiation and repair. In this study, we used Beas-2B human bronchial epithelial cells to investigate the effects of the TLR3 agonist polyinosinic:polycytidylic acid [Poly(I:C)] on airway cell migration and primary cilia (PC) formation. PC formation increased in cells incubated under serum deprivation. Migration was faster in Beas-2B cells pretreated with Poly(I:C) than in control cells, as judged by a wound healing assay, single-cell path tracking, and a Transwell migration assay. No changes in cell migration were observed when the cells were incubated in conditioned medium from Poly(I:C)-treated cells. PC formation was enhanced by Poly(I:C) treatment, but was reduced when the cells were exposed to the ciliogenesis inhibitor ciliobrevin A (CilioA). The inhibition of Beas-2B cell migration by CilioA was also assessed and a slight decrease in ciliogenesis was detected in SARS-CoV-2 spike protein (SP)-treated Beas-2B cells overexpressing ACE2 compared to control cells. Cell migration was decreased by SP but restored by Poly(I:C) treatment. Taken together, our results demonstrate that impaired migration by SP-treated cells can be attenuated by Poly(I:C) treatment, thus increasing airway cell migration through the regulation of ciliogenesis.

Improvement of osseointegration efficacy of titanium implant through plasma surface treatment.

A novel plasma treatment source for generating cylindrical plasma on the surface of titanium dental implants is developed herein. Using the titanium implant as an electrode and the packaging wall as a dielectric barrier, a dielectric barrier discharge (DBD) plasma was generated, allowing the implant to remain sterile. Numerical and experimental investigations were conducted to determine the optimal discharge conditions for eliminating hydrocarbon impurities, which are known to degrade the bioactivity of the implant. XPS measurement confirmed that plasma treatment reduced the amount of carbon impurities on the implant surface by approximately 60%. Additionally, in vitro experiments demonstrated that the surface treatment significantly improved cell adhesion, proliferation, and differentiation. Collectively, we proposed a plasma treatment source for dental implants that successfully removes carbon impurities and facilitate the osseointegration of SLA implants.

Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source.

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.

Enhanced osseointegration through direct energy deposition porous coating for cementless orthopedic implant fixation.

Direct energy deposition (DED) is a newly developed 3D metal printing technique that can be utilized on a porous surface coating of joint implants, however there is still a lack of studies on what advantages DED has over conventional techniques. We conducted a systematic mechanical and biological comparative study of porous coatings prepared using the DED method and other commercially available technologies including titanium plasma spray (TPS), and powder bed fusion (PBF). DED showed higher porosity surface (48.54%) than TPS (21.4%) and PBF (35.91%) with comparable fatigue cycle. At initial cell adhesion, cells on DED and PBF surface appeared to spread well with distinct actin stress fibers through immunofluorescence study. It means that the osteoblasts bind more strongly to the DED and PBF surface. Also, DED surface showed higher cell proliferation (1.27 times higher than TPS and PBF) and osteoblast cell activity (1.28 times higher than PBF) for 2 weeks culture in vitro test. In addition, DED surface showed better bone to implant contact and new bone formation than TPS in in vivo study. DED surface also showed consistently good osseointegration performance throughout the early and late period of osseointegration. Collectively, these results show that the DED coating method is an innovative technology that can be utilized to make cementless joint implants.

Erratum: Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source: publisher's note.

[This corrects the article on p. 5452 in vol. 12, PMID: 34692194.].

Osteo-Compatibility of 3D Titanium Porous Coating Applied by Direct Energy Deposition (DED) for a Cementless Total Knee Arthroplasty Implant: in Vitro and in Vivo Study.

: Direct energy deposition (DED) technology has gained increasing attention as a new implant surface technology that replicates the porous structure of natural bones facilitating osteoblast colonization and bone ingrowth. However, concerns have arisen over osteolysis or chronic inflammation that could be caused by Cobalt-chrome (CoCr) alloy and Titanium (Ti) nanoparticles produced during the fabrication process. Here, we evaluated whether a DED Ti-coated on CoCr alloy could improve osteoblast colonization and osseointegration in vitro and in vivo without causing any significant side effects. Three types of implant CoCr surfaces (smooth, sand-blasted and DED Ti-coated) were tested and compared. Three cell proliferation markers and six inflammatory cytokine markers were measured using SaOS2 osteoblast cells. Subsequently, X-ray and bone histomorphometric analyses were performed after implantation into rabbit femur. There were no differences between the DED group and positive control in cytokine assays. However, in the 5-bromo-2'-deoxyuridine (BrdU) assay the DED group exhibited even higher values than the positive control. For bone histomorphometry, DED was significantly superior within the 1000 µm bone area. The results suggest that DED Ti-coated metal printing does not affect the osteoblast viability or impair osseointegration in vitro and in vivo. Thus, this technology is biocompatible for coating the surfaces of cementless total knee arthroplasty (TKA) implants.

Emulating endothelial dysfunction by implementing an early atherosclerotic microenvironment within a microfluidic chip.

Recent studies on endothelial dysfunction in relation to vascular diseases including atherosclerosis have highlighted the key contribution of the microenvironment of endothelial cells (ECs). By mimicking the microenvironment of early atherosclerotic lesions, here, we replicate the pathophysiological phenotype and function of ECs within microchannels. Considering the elevated deposition of fibronectin (FN) in early atherosclerotic plaques and the close correlation between the vascular stiffness and the progression of atherosclerosis, we utilized FN coated hydrogels with increased stiffness for endothelial substrates within the microchannels. As a result, we demonstrated that endothelial integrity on FN coated microchannels is likely to be undermined exhibiting a random orientation in response to the applied fluid flow, notable disruption of vascular endothelial cadherins (VE-cadherins), and higher endothelial permeability as opposed to that on microchannels coated with collagen (CL), the atheroresistant vascular model.

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

Geometric constraints during epithelial jamming.

As an injury heals, an embryo develops, or a carcinoma spreads, epithelial cells systematically change their shape. In each of these processes cell shape is studied extensively whereas variability of shape from cell-to-cell is regarded most often as biological noise. But where do cell shape and its variability come from? Here we report that cell shape and shape variability are mutually constrained through a relationship that is purely geometrical. That relationship is shown to govern processes as diverse as maturation of the pseudostratified bronchial epithelial layer cultured from non-asthmatic or asthmatic donors, and formation of the ventral furrow in the Drosophila embryo. Across these and other epithelial systems, shape variability collapses to a family of distributions that is common to all. That distribution, in turn, is accounted for by a mechanistic theory of cell-cell interaction showing that cell shape becomes progressively less elongated and less variable as the layer becomes progressively more jammed. These findings suggest a connection between jamming and geometry that spans living organisms and inert jammed systems, and thus transcends system details. Although molecular events are needed for any complete theory of cell shape and cell packing, observations point to the hypothesis that jamming behavior at larger scales of organization sets overriding geometrical constraints.

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.