Marion H.M. Oudshoorn

The effect of photopolymerization on stem cells embedded in hydrogels.

Photopolymerizable hydrogels, formed by UV-exposure of photosensitive polymers in the presence of photoinitiators, are widely used materials in tissue engineering research employed for cellular entrapment and patterning. During photopolymerization, the entrapped cells are directly exposed to polymer and photoinitiator molecules. To develop strategies that prevent potential photoexposure-damage to osteoprogenitor cells, it is important to further characterize the effects of photopolymerization on the exposed cells. In this study we analyzed the viability, proliferation and osteogenic differentiation of multipotent stromal cell (MSC) monolayers after exposure to UV-light in the presence of Irgacure 2959, a frequently used photoinitiator in tissue engineering research. Cell cycle progression, apoptosis and osteogenic differentiation of encapsulated goat MSCs were studied in photopolymerized methacrylate-derivatized hyaluronic acid hydrogel and methacrylated hyperbranched polyglycerol gel. We demonstrate adverse effects of photopolymerization on viability, proliferation and reentry into the cell cycle of the exposed cells in monolayers, whereas the MSCs retain the ability to differentiate towards the osteogenic lineage. We further show that upon encapsulation in photopolymerizable hydrogels the viability of the embedded cells is unaffected by the photopolymerization conditions, while osteogenic differentiation depends on the type of hydrogel used.

Development of a murine model to evaluate the effect of vernix caseosa on skin barrier recovery

Abstract:  The aim of this study was twofold, that is the generation of a reliable model for skin barrier disruption and repair and to evaluate recovery of damaged skin after application of vernix caseosa (VC). VC was selected as its wound healing properties were suggested previously, but never clearly demonstrated. Five different levels of barrier disruption in mice, accomplished by tape‐stripping, were evaluated. Disruption models such as moderate, severe #1 and #2 (transepidermal water loss (TEWL) of 31 ± 2, 59 ± 4 and 66 ± 3 g/m2/h, respectively) showed complete recovery within 72 h. However, not all corneocytes were removed after tape‐stripping. Additionally, models such as severe #3 and #4 (TEWL of 73 ± 5 and 79 ± 6 g/m2/h, respectively) with a more severe disruption were evaluated. After tape‐stripping, all corneocytes were removed and the remaining epidermis was intact. However, model #3 still showed complete recovery within 72 h. With model #4, a crust was formed and almost complete recovery (approximately 90%) was obtained within only 8 days. The effect of VC application on recovery of disrupted skin was evaluated with model #3 and #4. Model #3 showed that application of VC predominantly influenced initial recovery and is therefore merely appropriate to study the effect of formulations in the initial recovery period. Topical application of VC on model #4 considerably increased initial and long‐term recovery. Moreover, VC application promoted rapid formation of stratum corneum and prevented epidermal thickening. These observations not only confirm the ability of VC to enhance barrier recovery, but also suggest potential use of this treatment clinically.

Lanolin-derived lipid mixtures mimic closely the lipid composition and organization of vernix caseosa lipids

The aim of the present study was to use semi-synthetic lipid mixtures to mimic the complex lipid composition, organization and thermotropic behaviour of vernix caseosa (VC) lipids. As VC shows multiple protecting and barrier supporting properties before and after birth, it is suggested that a VC substitute could be an innovative barrier cream for barrier deficient skin. Lanolin was selected as the source of the branched chain sterol esters and wax esters--the main lipid classes of VC. Different lipid fractions were isolated from lanolin and subsequently mixed with squalene, triglycerides, cholesterol, ceramides and fatty acids to generate semi-synthetic lipid mixtures that mimic the lipid composition of VC, as established by high-performance thin-layer chromatography. Differential scanning calorimetry and Fourier transform infrared spectroscopy investigations revealed that triglycerides play an important role in the (lateral) lipid organization and thermotropic behaviour of the synthetic lipid mixtures. Excellent resemblance of VC lipids was obtained when adding unsaturated triglycerides. Moreover, these lipid mixtures showed similar long range ordering as VC. The optimal lipid mixture was evaluated on tape-stripped hairless mouse skin in vivo. The rate of barrier recovery was increased and comparable to VC lipid treatment.

Skin barrier disruption by acetone: observations in a hairless mouse skin model

To disrupt the barrier function of the skin, different in vivo methods have been established, e.g., by acetone wiping or tape-stripping. In this study, the acetone-induced barrier disruption of hairless mice was investigated in order to establish a reliable model to study beneficial, long-term effects on barrier recovery after topical application. For both treatments (i.e., acetone treatment and tape-stripping) the transepidermal water loss directly after disruption and the subsequent barrier recovery profile were similar. Histological assessment showed significant lower number of corneocyte layers in acetone-treated and tape-stripped skin compared to untreated skin, while there was no statistical difference between the two treatments. Lipid analysis of acetone-treated skin revealed that only small fraction of lipids were extracted consisting of predominantly nonpolar lipids. Importantly, the ratio of the barrier lipids, i.e., cholesterol, free fatty acids and ceramides, remained similar between control and acetone-treated skin. This reflects the undisrupted lipid organization, as determined by small-angle X-ray diffraction measurements: the long-periodicity lamellar phase was still present after acetone treatment. Our results contradict earlier studies which reported no mechanical stratum corneum removal, a substantial extraction of lipids and disruption in lipid organization. In conclusion, our studies demonstrate that barrier disruption due to acetone treatment is mainly due to removal of corneocytes.

Mimicking vernix caseosa--preparation and characterization of synthetic biofilms.

The multiple protecting and barrier-supporting properties of the creamy, white biofilm vernix caseosa (VC) before and after birth suggest that a VC biomimetic could be an innovative barrier cream for barrier-deficient skin. The aim of this study was the rational design and preparation of synthetic biofilms mimicking the unique composition and properties of natural VC. Hexagonal, highly hydrated hyperbranched polyglycerol microgel particles (30 microm in diameter) were embedded in a synthetic lanolin-based lipid mixture using a micromixer. In these formulations, the water content of the particles (i.e. 50% and 80%), an additional lipid coating of the particles and different particle/lipid ratios were varied. Characterization with confocal laser scanning microscopy (CLSM) showed a homogeneous distribution of the labeled particles in the lipid matrix. Regarding structural appearance, particle density and distribution, the formulations with a high particle/lipid ratio (5:1) resembled native VC very closely. Comparable results between native VC and the synthetic formulations were obtained concerning water handling properties, thermotropic behavior while lower elasticity and lower viscosity were observed for the synthetic biofilms. The biofilm formulations were stable for at least 1 month at 4 degrees C. In conclusion, our formulations mimic natural VC very closely and are promising candidates for in vivo studies.

Effect of synthetic vernix biofilms on barrier recovery of damaged mouse skin

Abstract:  The aim of this work was to investigate whether topical application of synthetic biofilms supports and accelerates the recovery of the murine skin barrier, disrupted by sequential tape stripping. Therefore, various biofilms were applied topically on disrupted mouse skin to determine which formulation could improve barrier function, as was observed previously for the natural biofilm vernix caseosa (VC). The biofilms [i.e. particles (synthetic corneocytes) embedded in a synthetic lipid matrix] mimic closely the physicochemical properties and structure of VC. Various formulations were prepared using different particle:lipid ratios, particles with different initial water content and uncoated or lipid‐coated particles. It was observed that application of all tested formulations improved the skin barrier recovery rate and reduced crust formation and epidermal hyperproliferation. However, only one of the biofilms [i.e. B1; composed of uncoated particles with 50% (w/w) initial water content and particle:lipid ratio of 2:1] mimicked the effects of native VC most closely. This indicates the importance of the presence of individual components, i.e. barrier lipids and water, as well as the ratio of these components. Consequently, these observations suggest the potential use of this biofilm treatment clinically.