Closure Kinetics Research Articles

Thickness and Closure Kinetics of the Suprachoroidal Space Following Microneedle Injection of Liquid Formulations.

PurposeTo determine the effect of injection volume and formulation of a microneedle injection into the suprachoroidal space (SCS) on SCS thickness and closure kinetics.MethodsMicroneedle injections containing 25 to 150 μL Hanks' balanced salt solution (HBSS) were performed in the rabbit SCS ex vivo. Distribution of SCS thickness was measured by ultrasonography and three-dimensional (3D) cryo-reconstruction. Microneedle injections were performed in the rabbit SCS in vivo using HBSS, Discovisc, and 1% to 5% carboxymethyl cellulose (CMC) in HBSS. Ultrasonography was used to track SCS thickness over time.ResultsIncreasing HBSS injection volume increased the area of expanded SCS, but did not increase SCS thickness ex vivo. With SCS injections in vivo, the SCS initially expanded to thicknesses of 0.43 ± 0.06 mm with HBSS, 1.5 ± 0.4 mm with Discovisc, and 0.69 to 2.1 mm with 1% to 5% CMC. After injection with HBSS, Discovisc, and 1% CMC solution, the SCS collapsed to baseline with time constants of 19 minutes, 6 hours, and 2.4 days, respectively. In contrast, injections with 3% to 5% CMC solution resulted in SCS expansion to 2.3 to 2.8 mm over the course of 2.8 to 9.1 hours, after which the SCS collapsed to baseline with time constants of 4.5 to 9.2 days.ConclusionsWith low-viscosity formulations, SCS expands to a thickness that remains roughly constant, independent of the volume of fluid injected. Increasing injection fluid viscosity significantly increased SCS thickness. Expansion of the SCS is hypothesized to be controlled by a balance between the viscous forces of the liquid formulation and the resistive biomechanical forces of the tissue.

On the use of a four-cameras stereovision system to characterize large 3D deformation in elastomers

The mechanical behavior and volume change of filled elastomers were studied thanks to a four-cameras stereovision system. The device allows measuring simultaneously the displacement field on two faces of the sample using 3D Digital Image Correlation (3D DIC). Subset size, step size and filter size, associated with DIC calculations, are carefully calibrated to ensure efficient analysis of the displacement fields. The smoothing parameter (i.e. filter size times step size) appears to be a discriminating criterion with an upper limit below which the strain field can be appreciably estimated. Within the appropriate choice of analysis parameters, volume changes under large deformation can be evaluated. For sufficiently large deformation, volume change exhibits an upturn during stretching which could be the sign of a significant increase of void fraction. Volume change appears to be reversible during unloading phase when maximum stretch ratio is low enough. So cavities that could have been open under tension are closed when elastic deformation is released. Conversely, for high stretching ratio, volume change exhibits a hysteresis loop-like evolution indicating that either some plastic cavities remained or closure kinetics is slower.

Pereskia aculeata Miller leaves accelerate excisional wound healing in mice

Ethnopharmacological relevanceThe leaves of Pereskia aculeata Miller (Cactaceae), known as Barbados gooseberry, are used as emollients and to treat skin wounds and inflammatory process in Brazilian traditional medicine. Aim of the studyThis study investigated the topical wound healing activity of gels containing the methanol extract (ME) and hexane fraction (HF) of the leaves of this plant in a model of excisional wound healing in mice. Material and methodsMice were anesthetized and excisional skin wounds were performed using a circular metal punch of 5mm diameter. Next, the animals were treated with 30µL of topical gel formulations containing the gel base (vehicle), HF 5% or ME 5%. The treatments were applied immediately after the injury and every 48h during 14 days. To verify the wound closure kinetics, a digital caliper was used throughout this period. Laser Doppler perfusion image (LDPI) was applied to evaluate the blood flow rate at the injury site. Microscopic examination of the skin tissues was performed by histopathological analysis with hematoxylin and eosin and Gomori trichrome staining. Picrosirius-red staining was also used for morphometric analysis for collagen quantification. ResultsBoth HF and ME markedly accelerated the closeness of the skin wounds; however the HF activity was more evident, as this fraction induced the increase of blood flow rate and collagen deposition when statistically compared to the vehicle. The mice skin treated with HF and ME also showed less fibroplasia, blood vessels and inflammatory cells on the last day of experiment, which indicated a more advanced wound healing process. ConclusionsAs the wound healing process was considerably accelerated, especially by HF gel formulation, the results of this study not only contributed to better understand the ethnopharmacological application of P. acuelata leaves, but also encouraged further investigations on how to explore the potential uses of this plant in skin therapies.

Fibrin-based stem cell containing scaffold improves the dynamics of burn wound healing.

For severe burn injuries, successful medical intervention is accomplished by rapidly and safely providing physical barriers that can cover damaged skin tissues, thereby preventing critical danger of extensive bleeding and infection. Despite availability of a large assortment of wound coverage options, the etiology of wound healing is rather complex leading to significant defects in skin repair. The use of cell-mediated treatment approaches in combination with bioengineered wound coverage constructs may provide the missing tool to improve wound healing outcomes. In this study, we have used an engineered 3D PEGylated fibrin (P-fibrin) gel as a scaffold for adipose derived stem cells (ASCs) delivery into the burn injury model. We were able to confirm the presence of ASCs in the wound site two weeks after the initial injury. Delivery of ASCs-containing gels was associated with improved vascularization of the injured area at early time points accompanied by an increased abundance of mannose receptor expressing cells. Moreover, the application of P-fibrin biomaterial exhibited positive effects on early mononuclear cell recruitment and granulation tissue formation without negatively affecting wound closure kinetics or extent of connective tissue deposition. Collectively, our data support the feasibility of using P-fibrin gels in wound dressing applications requiring controlled delivery of viable cells.

Enhancement of Wound Healing by Non-Thermal N2/Ar Micro-Plasma Exposure in Mice with Fractional-CO2-Laser-Induced Wounds.

Micro-plasma is a possible alternative treatment for wound management. The effect of micro-plasma on wound healing depends on its composition and temperature. The authors previously developed a capillary-tube-based micro-plasma system that can generate micro-plasma with a high nitric oxide-containing species composition and mild working temperature. Here, the efficacy of micro-plasma treatment on wound healing in a laser-induced skin wound mouse model was investigated. A partial thickness wound was created in the back skin of each mouse and then treated with micro-plasma. Non-invasive methods, namely wound closure kinetics, optical coherence tomography (OCT), and laser Doppler scanning, were used to measure the healing efficiency in the wound area. Neo-tissue growth and the expressions of matrix metallopeptidase-3 (MMP-3) and laminin in the wound area were assessed using histological and immunohistochemistry (IHC) analysis. The results show that micro-plasma treatment promoted wound healing. Micro-plasma treatment significantly reduced the wound bed region. The OCT images and histological analysis indicates more pronounced tissue regrowth in the wound bed region after micro-plasma treatment. The laser Doppler images shows that micro-plasma treatment promoted blood flow in the wound bed region. The IHC results show that the level of laminin increased in the wound bed region after micro-plasma treatment, whereas the level of MMP-3 decreased. Based on these results, micro-plasma has potential to be used to promote the healing of skin wounds clinically.

Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands.

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

Cutting Edge: Regulatory T Cells Facilitate Cutaneous Wound Healing.

Foxp3-expressing regulatory T cells (Tregs) reside in tissues where they control inflammation and mediate tissue-specific functions. The skin of mice and humans contain a large number of Tregs; however, the mechanisms of how these cells function in skin remain largely unknown. In this article, we show that Tregs facilitate cutaneous wound healing. Highly activated Tregs accumulated in skin early after wounding, and specific ablation of these cells resulted in delayed wound re-epithelialization and kinetics of wound closure. Tregs in wounded skin attenuated IFN-γ production and proinflammatory macrophage accumulation. Upon wounding, Tregs induce expression of the epidermal growth factor receptor (EGFR). Lineage-specific deletion of EGFR in Tregs resulted in reduced Treg accumulation and activation in wounded skin, delayed wound closure, and increased proinflammatory macrophage accumulation. Taken together, our results reveal a novel role for Tregs in facilitating skin wound repair and suggest that they use the EGFR pathway to mediate these effects.

Micropore closure kinetics are delayed following microneedle insertion in elderly subjects

Transdermal delivery is an advantageous method of drug administration, particularly for an elderly population. Microneedles (MNs) allow transdermal delivery of otherwise skin-impermeable drugs by creating transient micropores that bypass the barrier function of the skin. The response of aging skin to MNs has not been explored, and we report for the first time that micropore closure is delayed in elderly subjects in a manner that is dependent upon MN length, number, and occlusion of the micropores. Twelve control subjects (25.6±2.8years) and 16 elderly subjects (77.3±6.8years) completed the study. Subjects were treated with MNs of 500μm or 750μm length, in arrays containing 10 or 50 MNs. Impedance measurements made at baseline, post-MN insertion, and at predetermined time points demonstrated that restoration of the skin barrier is significantly slower in elderly subjects under both occluded and non-occluded conditions. This was confirmed via calculation of the total permeable area created by the micropores (which would approximate the area available for drug delivery), as well as calculation of the micropore half-life. This pilot study demonstrates that longer timeframes are required to restore the barrier function of aged skin following MN insertion, suggesting that drug delivery windows could be longer following one treatment with a MN array.

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO2 in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO2 was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.

Myeloid Cell-Restricted Insulin/IGF-1 Receptor Deficiency Protects against Skin Inflammation.

Myeloid cells are key regulators of tissue homeostasis and disease. Alterations in cell-autonomous insulin/IGF-1 signaling in myeloid cells have recently been implicated in the development of systemic inflammation and insulin-resistant diabetes mellitus type 2 (DM). Impaired wound healing and inflammatory skin diseases are frequent DM-associated skin pathologies, yet the underlying mechanisms are elusive. In this study, we investigated whether myeloid cell-restricted IR/IGF-1R signaling provides a pathophysiologic link between systemic insulin resistance and the development of cutaneous inflammation. Therefore, we generated mice lacking both the insulin and IGF-1 receptor in myeloid cells (IR/IGF-1R(MKO)). Whereas the kinetics of wound closure following acute skin injury was similar in control and IR/IGF-1R(MKO) mice, in two different conditions of dermatitis either induced by repetitive topical applications of the detergent SDS or by high-dose UV B radiation, IR/IGF-1R(MKO) mice were protected from inflammation, whereas controls developed severe skin dermatitis. Notably, whereas during the early phase in both inflammatory conditions the induction of epidermal proinflammatory cytokine expression was similar in control and IR/IGF-1R(MKO) mice, during the late stage, epidermal cytokine expression was sustained in controls but virtually abrogated in IR/IGF-1R(MKO) mice. This distinct kinetic of epidermal cytokine expression was paralleled by proinflammatory macrophage activation in controls and a noninflammatory phenotype in mutants. Collectively, our findings provide evidence for a proinflammatory IR/IGF-1R-dependent pathway in myeloid cells that plays a critical role in the dynamics of an epidermal-dermal cross-talk in cutaneous inflammatory responses, and may add to the mechanistic understanding of diseases associated with disturbances in myeloid cell IR/IGF-1R signaling, including DM.

Kv3.1 uses a timely resurgent K(+) current to secure action potential repolarization.

High-frequency action potential (AP) transmission is essential for rapid information processing in the central nervous system. Voltage-dependent Kv3 channels play an important role in this process thanks to their high activation threshold and fast closure kinetics, which reduce the neuron's refractory period. However, premature Kv3 channel closure leads to incomplete membrane repolarization, preventing sustainable AP propagation. Here, we demonstrate that Kv3.1b channels solve this problem by producing resurgent K+ currents during repolarization, thus ensuring enough repolarizing power to terminate each AP. Unlike previously described resurgent Na+ and K+ currents, Kv3.1b's resurgent current does not originate from recovery of channel block or inactivation but results from a unique combination of steep voltage-dependent gating kinetics and ultra-fast voltage-sensor relaxation. These distinct properties are readily transferrable onto an orthologue Kv channel by transplanting the voltage-sensor's S3–S4 loop, providing molecular insights into the mechanism by which Kv3 channels contribute to high-frequency AP transmission.

Non-Markovian closure kinetics of flexible polymers with hydrodynamic interactions.

This paper presents a theoretical analysis of the closure kinetics of a polymer with hydrodynamic interactions. This analysis, which takes into account the non-Markovian dynamics of the end-to-end vector and relies on the preaveraging of the mobility tensor (Zimm dynamics), is shown to reproduce very accurately the results of numerical simulations of the complete nonlinear dynamics. It is found that Markovian treatments based on a Wilemski-Fixman approximation significantly overestimate cyclization times (up to a factor 2), showing the importance of memory effects in the dynamics. In addition, this analysis provides scaling laws of the mean first cyclization time (MFCT) with the polymer size N and capture radius b, which are identical in both Markovian and non-Markovian approaches. In particular, it is found that the scaling of the MFCT for large N is given by T ∼ N(3/2)ln(N/b(2)), which differs from the case of the Rouse dynamics where T ∼ N(2). The extension to the case of the reaction kinetics of a monomer of a Zimm polymer with an external target in a confined volume is also presented.

β-Structure within the Denatured State of the Helical Protein Domain BBL

Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.

Full-Thickness Thermal Injury Delays Wound Closure in a Murine Model.

Objective: The contemporary treatment of a full-thickness burn consists of early eschar excision followed by immediate closure of the open wound using autologous skin. However, most animal models study burn wound healing with the persistence of the burn eschar. Our goal is to characterize a murine model of burn eschar excision to study wound closure kinetics. Approach: C57BL/6 male mice were divided into three groups: contact burn, scald burn, or unburned control. Mice were burned at 80°C for 5, 10, or 20 s. After 2 days, the eschar was excised and wound closure was documented until postexcision day 13. Biopsies were examined for structural morphology and α-smooth muscle actin. In a subsequent interval-excision experiment (80°C scald for 10 s), the burn eschar was excised after 5 or 10 days postburn to determine the effect of a prolonged inflammatory focus. Results: Histology of both contact and scald burns revealed characteristics of a full-thickness injury marked by collagen coagulation and tissue necrosis. Excision at 2 days after a 20-s burn from either scald or contact showed significant delay in wound closure. Interval excision of the eschar, 5 or 10 days postburn, also showed significant delay in wound closure. Both interval-excision groups showed prolonged inflammation and increased myofibroblasts. Innovation and Conclusions: We have described the kinetics of wound closure in a murine model of a full-thickness burn excision. Both contact and scald full-thickness burn resulted in significantly delayed wound closure. In addition, prolonged interval-excision of the eschar appeared to increase and prolong inflammation.

Enhancement of Angiogenesis and Epithelialization Processes in Mice with Burn Wounds through ROS/RNS Signals Generated by Non‐Thermal N2/Ar Micro‐Plasma

Non‐thermal 0.5% N2/Ar micro‐plasma treatment with relatively high NO content was conducted on mice with second‐degree burn wounds. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations in the plasma‐exposed tissue lysate were measured. The wound closure kinetics, inflammatory responses, proliferation phase, blood flow, and formation of blood vessels in the mice were then assessed. The results showed that the wound contraction in the plasma‐exposed mice occurred five days earlier than that in the control group. The generated ROS/RNS signals stimulated the burn wound healing process, which were correlated with the angiogenesis and epithelialization processes. A possible in vivo mechanism for the enhancement of the processes in the plasma‐exposed mice is thereafter proposed.

Healable dual organic–inorganic crosslinked sol–gel based polymers: Crosslinking density and tetrasulfide content effect

ABSTRACTIn this article, the first generation of healable sol–gel based polymers is reported. A dual organic–inorganic crosslinked network is developed containing non‐reversible crosslinks and reversible (tetrasulfide) groups. The designed polymer architecture allows thermally induced mesoscale flow leading to damage closure followed by interfacial strength restoration due to reformation of the reversible groups. While the reversible bonds are responsible for the flow and the interface restoration, the irreversible crosslinks control the required mechanical integrity during the healing process. The temperature dependent gap closure kinetics is strongly affected by the crosslinking density and tetrasulfide content. Raman spectroscopy is used to explain the gap closure kinetics in air and dry nitrogen. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1953–1961

Hot isostatic pressing of single-crystal nickel-base superalloys: Mechanism of pore closure and effect on Mechanical properties

Pore annihilation was investigated in the single-crystal nickel-base superalloy CMSX-4. HIP tests at 1288 °C/103 MPa were interrupted at different times, then the specimens were investigated by TEM, metallography and density measurements. The kinetics of pore annihilation was determined. The pore closure mechanism was identified as plastic deformation on the octahedral slip systems. A model describing the kinetics of pore closure has been developed on the base of crystal plasticity and large strain theory. Mechanical tests with the superalloy CMSX-4 and the Ru-containing superalloy VGM4 showed, that HIP significantly increases the fatigue life at low temperatures but has no effect on creep strength.

Gravity affects the closure of the traps in Dionaea muscipula.

Venus flytrap (Dionaea muscipula Ellis) is a carnivorous plant known for its ability to capture insects thanks to the fast snapping of its traps. This fast movement has been long studied and it is triggered by the mechanical stimulation of hairs, located in the middle of the leaves. Here we present detailed experiments on the effect of microgravity on trap closure recorded for the first time during a parabolic flight campaign. Our results suggest that gravity has an impact on trap responsiveness and on the kinetics of trap closure. The possible role of the alterations of membrane permeability induced by microgravity on trap movement is discussed. Finally we show how the Venus flytrap could be an easy and effective model plant to perform studies on ion channels and aquaporin activities, as well as on electrical activity in vivo on board of parabolic flights and large diameter centrifuges.

Microneedle-Assisted Percutaneous Delivery of Naltrexone Hydrochloride in Yucatan Minipig: In Vitro–In Vivo Correlation

Although microneedle-assisted transdermal drug delivery has been the subject of multiple scientific investigations, very few attempts have been made to quantitatively relate in vitro and in vivo permeation. The case of naltrexone hydrochloride is not an exception. In the present study, a pharmacokinetic profile obtained following a "poke and patch" microneedle application method in the Yucatan minipig is reported. The profile demonstrates a rapid achievement of maximum naltrexone hydrochloride plasma concentration followed by a relatively abrupt concentration decline. No steady state was achieved in vivo. In an attempt to correlate the present in vivo findings with formerly published in vitro steady-state permeation data, a diffusion-compartmental mathematical model was developed. The model incorporates two parallel permeation pathways, barrier-thickness-dependent diffusional resistance, microchannel closure kinetics, and a pharmacokinetic module. The regression analysis of the pharmacokinetic data demonstrated good agreement with an independently calculated microchannel closure rate and in vitro permeation data. Interestingly, full-thickness rather than split-thickness skin employed in in vitro diffusion experiments provided the best correlation with the in vivo data. Data analysis carried out with the model presented herein provides new mechanistic insight and permits predictions with respect to pharmacokinetics coupled with altered microchannel closure rates.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.