Anisotropic dehydration of hydrogel surfaces
Efforts to develop tissue-engineered skin for regenerative medicine have explored natural, synthetic, and hybrid hydrogels. The creation of a bilayer material, with the stratification exhibited by native skin, is a complex problem. The mechanically robust, waterproof epidermis presents the stratum corneum at the tissue/air interface, which confers many of these protective properties. In this work, we explore the effect of high temperatures on alginate hydrogels, which are widely employed for tissue engineering due to their excellent mechanical properties and cellular compatibility. In particular, we investigate the rapid dehydration of the hydrogel surface which occurs following local exposure to heated surfaces with temperatures in the range 100–200 °C. We report the creation of a mechanically strengthened hydrogel surface, with improved puncture resistance and increased coefficient of friction, compared to an unheated surface. The use of a mechanical restraint during heating promoted differences in the rate of mass loss; the rate of temperature increase within the hydrogel, in the presence and absence of restraint, is simulated and discussed. It is hoped that the results will be of use in the development of processes suitable for preparing skin-like analogues; application areas could include wound healing and skin restoration.
KeywordsAlginate Dehydration Hydrogel Polysaccharide Skin Stratification
Biological tissues are highly organized and stratified structures. Complex tissue architectures are composed of different cell types with specific functions and locations. The interaction between them is conducted via their extracellular matrix. The interfaces between tissues are also complex and not easily distinguishable (Dormer et al. 2010). Tissue engineering aims to develop and mimic such architectures in vitro; various methods having been developed to simulate tissue complexity and allow interaction between different cell types, proteins, implanted materials, and scaffolds (Lee et al. 2009). Methods include 3D printing, electrospinning, and cell-sheeting engineering, often requiring complicated manipulations or lengthy constructions (Grossin et al. 2009).
Skin, the largest organ of the body, exhibits a stratified and organized structure, providing a protective layer with multiple functions (Horch et al. 2005). Nerve fibres and sensory receptors permit the detection of touch, pain, and temperature (Adams et al. 2007; Adams et al. 2013). There is significant interest in developing skin analogues suitable for replacing real skin, accelerating wound healing, restoring burns, and functioning like native skin (MacNeil 2007).
Skin tissue is composed of two layers: epidermis, a waterproof barrier that excludes microbes and retains body fluids; and dermis, beneath the epidermis, which is a collagen-rich connective tissue (Hunt et al. 2009). Melanocytes impart skin colour and are found at the lower level of the epidermis, and fibroblasts are found at the dermal layer and are responsible for the strength of the skin (MacNeil 2007; Bannasch et al. 2003). Epidermis, the outer skin layer, has a surface called stratum corneum, which is a less hydrated surface layer presented at the air/skin interface. Stratum corneum is composed of dead cells formed from keratin and with thickness that varies from 10 to 15 μm for humans (Johnson et al. 1993).
Significant progress has been made in the development of tissue-engineered skin (Shevchenko et al. 2010), the skin analogue produced using cells, extracellular matrix, and combinations thereof, although the use of autografts and allografts is associated with several limitations (Priya et al. 2008; Bello et al. 2001). Various methods have been utilized to achieve skin regeneration (Yang et al. 2000; Lechler and Fuchs 2005; Fuchs and Horsley 2008), or to manufacture skin products for wound healing. Most of the skin products that have been reported use a natural, synthetic, or hybrid hydrogel as a scaffold (Priya et al. 2008; Currie et al. 2001; Bakakrishnan et al. 2005; Boucard et al. 2007; Powell and Boyce 2009), for example the use of collagen matrix encapsulated with fibroblasts and seeded with keratinocytes (Yang et al. 2000). Due to the poor mechanical properties and difficulties of handling collagen hydrogel, hybrid collagen/alginate scaffolds have also been produced (Kim et al. 2011). Other hybrid scaffolds for skin regeneration include chitosan–gelatin bilayers (Mao et al. 2003). Stratified materials have also been produced using polyelectrolyte multilayers alternated with cell-containing gel layers (Grossin et al. 2009).
Alginate has been used in tissue engineering for the regeneration of skin, bone, and cartilage due to its mechanical properties and cellular compatibility, having been used to support the growth of fibroblasts and keratinocytes (Hunt et al. 2009; Kim et al. 2011; Alsberg et al. 2001; Stevens et al. 2004). Alginate is a naturally occurring, non-toxic polysaccharide derived from brown algae (Augst et al. 2006). It is biodegradable and can be used to form hydrogels under cytocompatible conditions. Alginate hydrogels are formed through ionotropic gelation of dissolved alginate in the presence of multivalent cations such as Mg2+, Ca2+, Sr2+, and Ba2+ (Morch et al. 2006; Topuz et al. 2012), and can be gelled using the internal (Chan et al. 2002a, b) or the external gelation method (Hunt et al. 2010; Kaklamani et al. 2014). Alginate has also been used to produce hybrid hydrogels for tissue engineering applications (Choi et al. 1999), but no solution proposed yet fulfils all the requirements needed to accomplish functional stratified structures.
The aim of this work was to investigate processes by which hydrogels can be modified to provide a stratum corneum-like dehydrated surface layer. Using a high-temperature heat source directly in contact with the hydrogel, the effect of rapid dehydration by evaporative loss of water from the hydrogel surface was studied. The influence of temperature and contact duration was considered. The external gelation method was employed to produce hydrogel samples; alginate concentration was also varied and investigated. The purpose of this research is to develop processes which might be suitable for the preparation of skin-like analogues.
All chemicals were sourced from Sigma Aldrich (UK) unless otherwise stated. Purities were > 99% in all cases. HPLC-grade H2O was employed throughout.
Hydrogels (HGs) were prepared using a previously reported external gelation method (Kaklamani et al. 2014). Briefly, sodium alginate (NaAlg) solution was poured into a poly(styrene) mould (141.4 mm inside diameter, 9.0 mm inside height, Sterilin, UK) to a liquid height of 6 mm and allowed to gel in the presence of an aqueous solution of CaCl2 held at the upper and lower boundaries by porous microcellulose sheets. Prior to the addition of the aqueous NaAlg solution, stainless steel cylindrical spacers (21 mm diameter, 6 mm height, Longshore Systems Engineering, UK) were placed at 60 o intervals around the inner edge of the mould to support the upper sheet. The volume of aqueous NaAlg solution required was 81.75 mL. The upper sheet was held in place from above using a poly(styrene) support, filled with water to maintain close contact between the upper sheet and the NaAlg solution as it gelled, since some shrinkage was observed at the sample edges.
Puncture testing was performed at 18 °C and 40% relative humidity using a Z030 mechanical tester (Zwick/Roell, UK). A stainless steel needle (gauge 19G, bevel point, Fisher Scientific, UK) was attached to a 5 N load cell. The puncture procedure involved approaching the needle towards the HG at a velocity of 5 mm/s. The specimen was oriented with the Dehydrated Surface presented upwards, facing the needle. Load–displacement data were recorded at 100 Hz.
Tribological measurements were performed on the Dehydrated Surface using a custom-built tribometer (Longshore Systems Engineering, UK). Tests employed a rotating stage travelling at 1 mm/s, on which the HG was securely immobilized. A sphere-on-flat contact geometry was adopted, employing 12.7 mm-diameter spheres made of (i) glass and (ii) poly(propylene). Measurements were performed using an open loop control system. The normal load was 0.49 ± 0.05 N for all tests.
Results and discussion
Restraining the samples during dehydration decreased the surface area from which water could evaporate. However, the metal disc was also heated during dehydration, by heat transfer through the HG. Hence, there was unrestricted heat and vapour loss at a solid/vapour interface for unrestrained samples, but this is not true for samples which were restrained. The thickness of the dehydrated layer increases with increasing temperature, in accordance with expectations. It should be noted that exposure to temperatures higher than 200 °C tended to lead to a burnt HG surface.
Tribological testing versus glass and polypropylene
Friction coefficient of cauterized alginate against glass and poly(propylene)
Friction coefficient vs glass
Friction coefficient vs poly(propylene)
0.31 ± 0.01
0.12 ± 0.01
Dehydrated, 200 °C, 4 min
0.63 ± 0.02
0.28 ± 0.01
Effect of restraint on heating rate
Parameters used for heat transfer simulations
ρ (kg m−3)
Cp (kJ kg−1 K−1)
k (W m−1 K−1)
Aluminium hot platea
Considering the E = 189 kPa HGs in Fig. 3, at T = 100 °C the restrained HG loses less mass than the unrestrained HG. This is because the temperature of the restrained HG increases more slowly than the unrestrained HG. The HG temperature does not reach 100 °C, the boiling point of water, for either the restrained or unrestrained condition. At T = 150 and 200 °C, the restrained HGs exhibit greater mass loss than the unrestrained HGs; why might this be? Here, the HG temperature rises to an excess of 100 °C for both the restrained and unrestrained conditions. It is suggested that energy retained within the restrained HG/aluminium disc increases the rate of water evaporation, which otherwise would have caused an increase in temperature. In comparison, this energy is conducted through the unrestrained HG and is lost at the HG/air interface. The difference in behaviour occurs when the hot plate temperature is increased above the boiling point of water.
We report the effect of local exposure to high temperatures on the surface properties of alginate hydrogels. Rapid dehydration of the surface using temperatures in the range 100–200 °C leads to the creation of a mechanically strengthened hydrogel surface layer, exhibiting improved puncture resistance and increased coefficient of friction compared to the unheated surface. Direct contact with a surface at 200 °C for 4 min produced the most mechanically robust layer. Mechanical constraint of the hydrogel sample was important during processing, preventing curvature of the sample during cauterization, particularly at the edges.
Further investigations will study a greater variety of HG compositions, as well as explore in more detail the importance of dehydration temperature, duration, the thermal conductivity of the HG, and the nature of the restraint material. A modified apparatus for dehydration within a humidified atmosphere, for reducing evaporative loss, is currently being designed. The possible benefit of performing the heating whilst the HG is immersed in an aqueous solution, perhaps a physiologically relevant buffer, is also under consideration. Finally, an improved understanding of the material structure remaining following the dehydration process would be useful.
This work was supported by the European Union under the FP7 programme (NANOBIOTOUCH Project: FP7-NMP-228844). The Zwick/Roell Z030 mechanical tester and Longshore Systems Engineering tribometer used in this research were obtained through Birmingham Science City: Innovative Uses for Advanced Materials in the Modern World (West Midlands Centre for Advanced Materials Project 2), with support from Advantage West Midlands (AWM) and partly funded by the European Regional Development Fund (ERDF). The authors thank the assistance of Elaine Mitchell throughout the course of this research.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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