The Reliability of PCL/Anti-VEGF Electrospun Scaffolds to Support Limbal Stem Cells for Corneal Repair.

Since only few reported studies propose anti-vascular endothelial growth factor (anti-VEGF) delivery through electrospun scaffolds, this study greatly contributes to the potential prevention of patient's vision loss, as it explores electrospun polycaprolactone (PCL) coated with anti-VEGF for the blockage of abnormal cornea vascularization. In terms of physicochemical properties, the biological component increased the PCL scaffold fiber diameter (by ~24%) and pore area (by ~82%), while ut slightly reduced its total porosity as the anti-VEGF solution filled the voids of the microfibrous structure. The addition of the anti-VEGF increased the scaffold stiffness almost three-fold at both strains of 5 and 10%, as well as its biodegradation rate (~36% after 60 days) with a sustained release profile after Day 4 of phosphate buffered saline incubation. In terms of scaffold application function, the PCL/Anti-VEGF scaffold proved to be more favorable for the adhesion of cultured limbal stem cells (LSCs); this was confirmed by the SEM images, where the cells showed flat and elongated conformations. Further support of the LSC growth and proliferation was confirmed by the identified p63 and CK3 markers after cell staining. These results demonstrate the advantageous effect of the surface-adsorbed anti-VEGF to stop vision loss and help damaged corneal tissue repair.

Detection of Limbal Stem Cells Adhered to Melt Electrospun Silk Fibroin and Gelatin-Modified Polylactic Acid Scaffolds.

Limbal stem cells (LSCs) are of paramount importance in corneal epithelial tissue repair. The cornea becomes opaque in case of limbal stem cell deficiency (LSCD), which may cause serious damage to the ocular visual function. There are many techniques to restore damaged epithelium, one of which is the transplantation of healthy cultured LSCs, usually onto a human amniotic membrane or onto bio-based engineered scaffolds in recent years. In this study, melt electrospun polylactic acid (PLA) was modified by silk fibroin or gelatin and further cultured with LSCs originating from three different donors. In terms of physicochemical properties, both modifications slightly increased PLA scaffold porosity (with a significantly larger pore area for the PLA/gelatin) and improved the scaffolds' swelling percentage, as well as their biodegradation rate. In terms of the scaffold application function, the aim was to detect/visualize whether LSCs adhered to the scaffolds and to further determine cell viability (total number), as well as to observe p63 and CK3 expressions in the LSCs. LSCs were attached to the surface of microfibers, showing flattened conformations or 3D spheres in the formation of colonies or agglomerations, respectively. All scaffolds showed the ability to bind the cells onto the surface of individual microfibers (PLA and PLA/gelatin), or in between the microfibers (PLA/silk fibroin), with the latter showing the most intense red fluorescence of the stained cells. All scaffolds proved to be biocompatible, while the PLA/silk fibroin scaffolds showed the highest 98% viability of 2.9 × 106 LSCs, with more than 98% of p63 and less than 20% of CK3 expressions in the LSCs, thus confirming the support of their growth, proliferation and corneal epithelial differentiation. The results show the potential of these bio-engineered scaffolds to be used as an alternative clinical approach.

Bioactivity Comparison of Electrospun PCL Mats and Liver Extracellular Matrix as Scaffolds for HepG2 Cells.

Cells grown on bioactive matrices have immensely advanced many aspects of biomedical research related to drug delivery and tissue engineering. Our main objective was to perform simple evaluation of the structural and biotic qualities of cell scaffolds made of affordable biomaterials for liver cell line (HepG2) cultivation in vitro. In this work the electrospun matrix made of synthetic polyester poly(ε-caprolactone) (PCL) was compared with the natural protein-based extracellular matrix isolated from porcine liver (ECM). Mechanical and structural analysis showed that ECM was about 12 times less resistant to tensile stress while it had significantly larger pore size and twice smaller water contact angle than PCL. Bioactivity assessment included comparison of cell growth and transfection efficiency on cell-seeded scaffolds. Despite the differences in composition and structure between the two respective matrices, the rate of cell spreading and the percentage of transfected cells on both scaffolds were fairly comparable. These results suggest that in an attempt to produce simple, cell carrying structures that adequately simulate the natural scaffold, one can rely on PCL electrospun mats.

Poly(ε-caprolactone) Titanium Dioxide and Cefuroxime Antimicrobial Scaffolds for Cultivation of Human Limbal Stem Cells.

Limbal Stem Cell Deficiency (LSCD) is a very serious and painful disease that often results in impaired vision. Cultivation of limbal stem cells for clinical application is usually performed on carriers such as amniotic membrane or surgical fibrin gel. Transplantation of these grafts is associated with the risk of local postoperative infection that can destroy the graft and devoid therapeutic benefit. For this reason, electrospun scaffolds are good alternatives, as proven to mimic the natural cells surroundings, while their fabrication technique is versatile with regard to polymer functionalization and scaffolds architecture. This study considers the development of poly(ε-caprolactone) (PCL) immune-compatible and biodegradable electrospun scaffolds, comprising cefuroxime (CF) or titanium dioxide (TiO2) active components, that provide both bactericidal activity against eye infections and support of limbal stem cells growth in vitro. The PCL/CF scaffolds were prepared by blend electrospinning, while functionalization with the TiO2 particles was performed by ultrasonic post-processing treatment. The fabricated scaffolds were evaluated in regard to their physical structure, wetting ability, static and dynamic mechanical behaviour, antimicrobial efficiency and drug release, through scanning electron microscopy, water contact angle measurement, tensile testing and dynamic mechanical analysis, antimicrobial tests and UV-Vis spectroscopy, respectively. Human limbal stem cells, isolated from surgical remains of human cadaveric cornea, were cultured on the PCL/CF and PCL/TiO2 scaffolds and further identified through immunocytochemistry in terms of cell type thus were stained against p63 marker for limbal stem cells, a nuclear transcription factor and cytokeratin 3 (CK3), a corneal epithelial differentiation marker. The electrospun PCL/CF and PCL/TiO2 successfully supported the adhesion, proliferation and differentiation of the cultivated limbal cells and provided the antimicrobial effect against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.

Synthetic vs natural scaffolds for human limbal stem cells.

AIM: To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. METHODS: Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. RESULTS: Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. CONCLUSION: Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes.

Multiscale phenomena related to diabetic foot.

Diabetes is a group of metabolic diseases causing a system disorder, i.e.; it cannot be explained or understood by phenomena on single material scale. The diabetic foot is studied as flexible multibody structure by nonlinear finite element method. The physical and geometrical multiscale heterogeneity is solved by multilevel finite element approach. The diabetic tissue is described by internal coordinate's formalism, as complex multiscale process in tissue. The accompanying problem of the axisymetric wound healing is solved numerically. Some results related to foot deformity, stress and strain concentration and wound healing are presented.

The comparison of human perception to the measured sweat transfer rate.

The subjective investigation, that is focused on the sensations of a person, is a good tool for the evaluation of an environment that group of people consider comfortable. In the experiment reported here, participants were dressed into 1-layer and 2-layer clothing systems. They performed physical activity and rated the subjective perception of comfort, as well as the thermal and moisture sensation. The aim of this investigation is to compare the subjective human perception during the physical activity wearing different clothing systems to the objective results of sweat absorption.

Comparison of subjective and objective measurement of sweat transfer rate.

Sweat absorption is investigated using the subjective and objective methods. The subjective investigation analyses the physiological responses of male and female volunteers wearing two kinds of textile fabrics (cotton and polyester) during certain physical activity. The subjects were exposed to the different conditions of ambient temperature (23, 26, 29 and 32 degrees C) and constant relative humidity of 67 +/- 3%. In the objective investigation the sweating guarded hotplate (SGHP) system was used that stimulates the processes of sweat transfer between the human skin, textile material and environment. The results of mass absorption obtained from the objective measurement on the sweating guarded hotplate show the similar trend as the results obtained during the subjective measurement.

Biomechanical model of the diabetic foot.

In this work, a two dimensional (2D) finite element foot model was established from magnetic resonance imaging (MRI) of a male subject. The model comprises first medial planar cross-section through the foot, representing the foot in standing posture. For specified external load, the stress and strain distribution field under foot structure are determined. The material characterization of foot structure components are stronger related to diabetic phenomena. The new material model for soft tissue based on mixture theory is proposed. The linear finite element model replaced by nonlinear counterpart with segment-to-segment contact element.

A case study of Chinese bound feet: application of footprint analysis.

Foot print patterns of the bound feet of a 90-year-old Chinese female were made to obtain insight into the ergonomic consequences of a Chinese custom that caused significant disabilities for many women throughout history. Pressure patterns were evaluated using the techniques applied to standard thumb print analsyis. A digital summary of the pressure patterns were compared to the patterns obtained from a normal subject. The outcomes indicated that the bound foot produced greater plantar tissue pressures than the non-bound foot. These observations help explain the discomfort, gait abnormalities, and disabilities exhibited by many older women with bound feet living in China today. Although foot-binding is no longer practiced, this study offers an ergonomic perspective on a custom practiced in China for centuries.

Blood flow multiscale phenomena.

The cardiovascular disease is one of most frequent cause deaths in modern society. The objective of this work is analyse the effect of dynamic vascular geometry (curvature, torsion, bifurcation) and pulsatile blood nature on secondary flow, wall shear stress and platelet deposition. The problem was examined as multi-scale physical phenomena using perturbation analysis and numerical modelling. The secondary flow determined as influence pulsatile pressure, vascular tube time-dependent bending and torsion on the main axial flow. Bifurcation and branching phenomena are analysed experimentally through, blood-like fluid pulsatile flow across elastic rubber-like Y-model model. The problem complex geometry near branching in platelet deposit modelling is resolved numerically as Falker-Skan flow.

The cancellous bone multiscale morphology-elasticity relationship.

The cancellous bone effective properties relations are analysed on multiscale across two aspects; properties of representative volume element on micro scale and statistical measure of trabecular trajectory orientation on mesoscale. Anisotropy of the microstructure is described across fabric tensor measure with trajectory orientation tensor as bridging scale connection. The scatter measured data (elastic modulus, trajectory orientation, apparent density) from compression test are fitted by stochastic interpolation procedure. The engineering constants of the elasticity tensor are estimated by last square fitt procedure in multidimensional space by Nelder-Mead simplex. The multiaxial failure surface in strain space is constructed and interpolated by modified super-ellipsoid.

Foot anthropometry and morphology phenomena.

Foot structure description is important for many reasons. The foot anthropometric morphology phenomena are analyzed together with hidden biomechanical functionality in order to fully characterize foot structure and function. For younger Croatian population the scatter data of the individual foot variables were interpolated by multivariate statistics. Foot structure descriptors are influenced by many factors, as a style of life, race, climate, and things of the great importance in human society. Dominant descriptors are determined by principal component analysis. Some practical recommendation and conclusion for medical, sportswear and footwear practice are highlighted.

Experimental flow studies in an elastic Y-model.

To determine the causes and history of atherosclerosis it is necessary to understand the hemodynamic parameters of blood circulation. Hemodynamic parameters play an important role in the formation of atherosclerotic plaques, especially near bends and bifurcations where the flow separates from the wall. Here the flow is laminar and non-axial with eddies, secondary flow, flow separation and stagnation points. Stenoses are found predominantly in flow separation areas. Therefore, it is important to separately study the following flow parameters: steady and pulsatile flow, wall elasticity and non-Newtonian flow behavior of blood. A simplified silicon elastic y-model simulating the human carotid artery was used for the analysis of these parameters. This model can be used for numerical studies as well. Flow was visualized at steady flow using dyes and at pulsatile flow with a photoelastic apparatus and a birefringent solution. The local axial velocity at steady and pulsatile flow was determined with a one-component Laser-Doppler-Anemometer (LDA). Pulsatile flow was generated by a piston membrane pump. A glycerin-water solution was used to simulate the Newtonian flow behavior of blood. A DMSO-Separan water solution was used to simulate the non-Newtonian flow behavior. Pulsatile flow creates higher and lower shear rates so called oscillating shear rate compare to steady flow depending on the velocity amplitude. The non-Newtonian fluid showed a markedly different flow behavior than the Newtonian fluid especially in areas of flow separation. Shear gradients were calculated from these velocity measurements using a bicubic spline interpolation. Shear stresses were calculated from these velocity shear gradients and the viscosity of the non-Newtonian fluid at these shear gradients. At special areas, high shear stresses > 10 Pa were found. The elasticity of the model wall also influences the flow behavior. The measurements showed that the characteristics of pulsatile flow and the elasticity of the model wall should be observed concomitantly. This paper presents the steady and pulsatile flow with a Newtonian and non-Newtonian fluid in an elastic model.