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PURPOSE\nThe aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT).\nMETHODS\nA novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization.\nRESULTS\nThe applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 μm), -volumes (1-100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold).\nCONCLUSION\nIntradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Background Repositioning of individuals with reduced mobility and at risk of pressure ulcers is an essential preventive step. Manual or automatic lateral tilting is a way of doing this and the international guidelines propose a 30° to 40° side lying position. The goal of the present study was to determine the internal strains in individuals lying in a supine position and during tilting. Methods Based on magnetic resonance imaging (MRI) of the sacral area of human volunteers, subject specific finite element models were developed. By comparing calculated contours of the skin, fat and muscle with MRI measurements on a flat surface the models were validated. A parameter study was performed to assess the sensitivity of the model for changes in material properties. Simulations were performed at tilting angles of volunteers between 0° and 45°. Findings Subjects in a supine position or tilted have the highest strains in the muscle and fat. Tilting does affect the strain distribution, taking away the highest peak strains. There seems to exist an optimal tilting angle between 20° and 30°, which may vary depending on factors such as BMI of the subject and is in the current paper investigated only for the sacrum. Interpretation The study shows that tilting indeed has a significant, positive influence on internal strains, which is important for the prevention of deep tissue injury. Additional studies are needed to draw conclusions about the greater trochanter area and the tissues around the shoulder.

Deep tissue injury (DTI), a type of pressure ulcer, arises in the muscle layers adjacent to bony prominences due to sustained mechanical loading. DTI presents a serious problem in the clinic, as it is often not visible until reaching an advanced stage. One of the causes can be direct mechanical deformation of the muscle tissue and cell. The mechanism of cell death induced by mechanical compression was studied using bio-artificial skeletal muscle tissues. Compression was applied by placing weights on top of the constructs. The morphological changes of the cytoskeleton and the phosphorylation of mitogen-activated protein kinases (MAPK) under compression were investigated. Moreover, inhibitors for each of the three major MAPK groups, p38, ERK, and JNK, were applied separately to look at their roles in the compression caused apoptosis and necrosis. The present study for the first time showed that direct mechanical compression activates MAPK phosphorylation. Compression also leads to a gradual destruction of the cytoskeleton. The percentage apoptosis is strongly reduced by p38 and JNK inhibitors down to the level of the unloaded group. This phenomenon could be observed up to 24. h after initiation of compression. Therefore, cell death in bio-artificial muscle tissue caused by mechanical compression is primarily caused by a physiological mechanism, rather than through a physical mechanism which kills the cell directly. These findings reveal insight of muscle cell death under mechanical compression. Moreover, the result indicates a potential clinical solution to prevent DTI by pre-treating with p38 or/and JNK inhibitors.

The skin is a promising location for vaccination with its abundant population ofantigen capturing and presenting cells. The development of new techniques, suchas the use of microneedles, can facilitate the delivery of vaccines into theskin. In recent years, many different types of microneedle arrays have beendesigned. However, their geometry and arrangement within an array may beoptimized to trigger sufficient antigen presenting cells. A computational modelcan support the rational design of microneedle arrays. Therefore, the aim of thecurrent study was to describe the distribution and kinetics of a deliveredantigen within the skin using a theoretical compartment model, which includedbinding of antigens to receptors and their uptake by cells, and to determinewhich parameters should be measured to validate the model for a specificapplication. Multiple simulations were performed using a high and low antigendelivery dose and a range of values for the rate constants. The resultsindicated that the cells were highly saturated when a high dose was applied,while for a low dose saturation was only reached in 5% of the simulations. Thiswas caused by the difference in the ratio between the administered dose and theavailable binding sites and suggests the dose should be adapted to the number ofcells and receptors for a specific compound. The sensitivity analysis of themodel parameters confirmed that the initial dose and receptor concentrationswere indeed the two parameters that had the largest influence on the variance inantigen concentrations within the cells and circulation at equilibrium. Hence,these parameters are important to be measured in vivo. The presentedpharmacokinetics model can be used in future computational models to predict theinfluence of microneedle array geometry to optimize their design.

Understanding collagen and stress fiber remodeling is essential for the development of engineered tissues with good functionality. These processes are complex, highly interrelated, and occur over different time scales. As a result, excessive computational costs are required to computationally predict the final organization of these fibers in response to dynamic mechanical conditions. In this study, an analytical approximation of a stress fiber remodeling evolution law was derived. A comparison of the developed technique with the direct numerical integration of the evolution law showed relatively small differences in results, and the proposed method is one to two orders of magnitude faster.

The anisotropic collagen architecture of an engineered cardiovascular tissue has a major impact on its in vivo mechanical performance. This evolving collagen architecture is determined by initial scaffold microstructure and mechanical loading. Here, we developed and validated a theoretical and computational microscale model to quantitatively understand the interplay between scaffold architecture and mechanical loading on collagen synthesis and degradation. Using input from experimental studies, we hypothesize that both the microstructure of the scaffold and the loading conditions influence collagen turnover. The evaluation of the mechanical and topological properties of in vitro engineered constructs reveals that the formation of extracellular matrix layers on top of the scaffold surface influences the mechanical anisotropy on the construct. Results show that the microscale model can successfully capture the collagen arrangement between the fibers of an electrospun scaffold under static and cyclic loading conditions. Contact guidance by the scaffold, and not applied load, dominates the collagen architecture. Therefore, when the collagen grows inside the pores of the scaffold, pronounced scaffold anisotropy guarantees the development of a construct that mimics the mechanical anisotropy of the native cardiovascular tissue.

In order to maintain tissue homeostasis and functionality, adherent cells need to sense and respond to environmental mechanical stimuli. An important ability that adherent cells need in order to properly sense and respond to mechanical stimuli is the ability to exert contractile stress onto the environment via actin stress fibers. The actin stress fibers form a structural chain between the cells' environment via focal adhesions and the nucleus via the nuclear lamina. In case one of the links in this chain is missing or aberrant, contractile stress generation will be affected. This is especially the case in laminopathic cells, which have a missing or mutated form of the LMNA gene encoding for part of the nuclear lamina. Using the thin film method combined with sample specific finite element modeling, we quantitatively showed a fivefold lower contractile stress generation of Lmna knockout mouse embryonic fibroblasts (MEFs) as compared to wild-type MEFs. Via fluorescence microscopy it was demonstrated that the lower contractile stress generation was associated with an impaired actin stress fiber organization with thinner actin fibers and smaller focal adhesions. Similar experiments with wild-type MEFs with chemically disrupted actin stress fibers verified these findings. These data illustrate the importance of an organized actin stress fiber network for contractile stress generation and demonstrate the devastating effect of an impaired stress fiber organization in laminopathic fibroblasts. Next to this, the thin film method is expected to be a promising tool in unraveling contractility differences between fibroblasts with different types of laminopathic mutations.

Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH 5.8 and nearly uncharged at pH 7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ± 0.6 μg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5 μg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization.

Microneedle arrays have been developed to deliver a range of biomolecules including vaccines into the skin. These microneedles have been designed with a wide range of geometries and arrangements within an array. However, little is known about the effect of the geometry on the potency of the induced immune response. The aim of this study was to develop a computational model to predict the optimal design of the microneedles and their arrangement within an array. The three-dimensional finite element model described the diffusion and kinetics in the skin following antigen delivery with a microneedle array. The results revealed an optimum distance between microneedles based on the number of activated antigen presenting cells, which was assumed to be related to the induced immune response. This optimum depends on the delivered dose. In addition, the microneedle length affects the number of cells that will be involved in either the epidermis or dermis. By contrast, the radius at the base of the microneedle and release rate only minimally influenced the number of cells that were activated. The model revealed the importance of various geometric parameters to enhance the induced immune response. The model can be developed further to determine the optimal design of an array by adjusting its various parameters to a specific situation.

The purpose of this study was to investigate the effect of various repeated fractional intradermal dosing schedules of inactivated polio vaccine serotype 1 (IPV1) on IPV1-specific IgG responses in rats. By utilizing an applicator that allowed for precisely controlled intradermal microinjections by using a single hollow microneedle, rats were immunized intradermally with 5 D-antigen units (DU) of IPV1 at 150μm skin depth. This dose was administered as a bolus, or in a repeated fractional dosing schedule: 4 doses of 1.25 DU (1/4th of total dose) were administered on four consecutive days or every other day; 8 doses of 0.625 DU (1/8th of total dose) were administered on eight consecutive days; or 4 exponentially increasing doses (0.04, 0.16, 0.8 and 4 DU), either with or without an exponentially increasing CpG oligodeoxynucleotide 1826 (CpG) dose, were administered on four consecutive days. All of these fractional dosing schedules resulted in up to ca. 10-fold higher IPV1-specific IgG responses than intradermal and intramuscular bolus dosing. IPV1 combined with adjuvant CpG in exponential dosing did not significantly increase the IPV1-specific IgG responses further, which demonstrated that maximal responses were achieved by fractional dosing. In conclusion, repeated fractional intradermal IPV1 dosing leads to superior IPV1-specific IgG responses without the use of adjuvants. These results indicate that a controlled release delivery system for intradermal IPV1 delivery can potentiate IPV1-specific IgG responses.

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm(2)/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin.

The paper describes the current views on the cause of a sub-class of pressure ulcers known as pressure induced deep tissue injury (DTI). A multi-scale approach was adopted using model systems ranging from single cells in culture, tissue engineered muscle to animal studies with small animals. This has led to a clear understanding on two damage mechanisms associated with the development of DTI. Direct deformation results from high, but physiologically relevant, strains and is a process that leads to the first signs of cell damage within minutes. Ischaemic damage is caused by occlusion of blood vessels, but takes several hours to develop. The paper ends with some clinical consequences.