Skin layer mechanics

Skin layer mechanics

Citation for published version (APA):

Geerligs, M. (2010). Skin layer mechanics. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR657803

DOI:

10.6100/IR657803

Document status and date: Published: 01/01/2010

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Skin layer mechanics

ISBN: 978-90-74445-92-4

Cover design: Marion Geerligs & Henny Herps

Printed by Universiteitsdrukkerij TU Eindhoven, Eindhoven, The Netherlands.

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Skin layer mechanics

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de

Technische Universiteit Eindhoven, op gezag van de

rector magnificus, prof.dr.ir. C.J. van Duijn, voor een

commissie aangewezen door het College voor

Promoties in het openbaar te verdedigen

op donderdag 21 januari 2010 om 16.00 uur

door

Marion Geerligs

Dit proefschrift is goedgekeurd door de promotor:

prof.dr.ir. F.P.T. Baaijens

Copromotoren:

dr.ir. C.W.J. Oomens

en

dr.ir. G.W.M. Peters

Contents

Summary ... ix

Skin layer mechanics ... ix

Chapter 1 General introduction ... 1

1.1 Introduction ... 2

1.2 A mechanical view of skin anatomy and physiology ... 4

1.2.1 Skin topography ... 4 1.2.2 Stratum corneum ... 5 1.2.3 Viable epidermis ... 6 1.2.4 Dermal-epidermal junction ... 7 1.2.5 Dermis... 8 1.2.6 Hypodermis... 9

1.3 Review of skin layer mechanics ... 10

1.3.1 In vivo vs in vitro experiments ... 10

1.3.2 Mechanical behavior of the stratum corneum ... 10

1.3.3 Mechanical behavior of the viable epidermis ... 12

1.3.4 Hypodermis... 12

1.4 Aim and Outline ... 13

Chapter 2 Isolation and preservation methods for the epidermis

and stratum corneum ... 15

2.1 Introduction ... 16

2.2 Skin preparation and analyses ... 17

2.2.1 Skin preparation ... 17

2.2.2 Histological examination ... 18

2.2.3 Analyses of skin viability ... 19

2.3.1 Mechanical separation ... 19

2.3.2 Ionic change ... 20

2.3.3 Heat ... 21

2.3.4 Enzymatic digestion ... 21

2.3.5 Microwave irradiation ... 23

2.4 Isolation techniques for the stratum corneum... 23

2.4.1 Mechanical separation ... 24

2.4.2 Chemical separation ... 25

2.4.3 Enzymatic digestion ... 25

2.5 Preservation of the upper skin layers ... 26

2.5.1 Short-term storage ... 27

2.5.2 Long-term storage ... 28

2.6 Discussion ... 30

Chapter 3 Linear shear response of the upper skin layers ... 33

3.1 Introduction ... 34 3.2 Methods ... 35 3.2.1 Sample preparation ... 35 3.2.2 Experimental set-up ... 36 3.2.3 Rheological methods ... 39 3.2.4 Experimental procedures ... 40 3.3 Results ... 41 3.4 Discussion ... 46

Chapter 4 A new indentation method to determine mechanical

properties of the epidermis ... 49

4.1 Introduction ... 50

4.1.1 Sample preparation ... 51

4.1.2 Experimental procedure ... 53

4.1.3 Determination of the Young‟s modulus ... 54

4.2 Results ... 55

4.3 Discussion ... 56

Chapter 5 Linear viscoelastic behavior of subcutaneous adipose

tissue ... 61

5.1 Introduction ... 62

5.2 Methods and Materials ... 64

5.2.1 Sample preparation ... 64

5.2.2 Rheological methods ... 64

5.2.3 Testing procedure ... 65

5.2.4 Statistics ... 66

5.3.1 Small oscillatory strain behavior ... 67

5.3.2 Model application ... 68

5.3.3 Time-Temperature Superposition ... 69

5.3.4 Freezing effects ... 70

5.4 Discussion ... 71

Chapter 6 Does subcutaneous adipose tissue behave as an

(anti-)thyxotropic material? ... 73

6.1 Introduction ... 74

6.2 Materials & Methods ... 75

6.2.1 Sample preparation ... 75

6.2.2 Rheological methods ... 76

6.3 Results ... 78

6.3.1 Long term small strain behavior ... 78

6.3.2 Large strain experiments ... 79

6.4 Discussion ... 81

Chapter 7 General discussion ... 85

7.1 Introductory remarks ... 86

7.2 In vitro model ... 87

7.3 Mechanical methods ... 88

7.4 Main findings ... 90

7.4.1 Small strain behavior of the epidermal layers ... 90

7.4.2 Mechanical behavior of the subcutaneous adipose tissue ... 91

7.5 Implications for clinical and cosmetic applications ... 91

7.6 Recommendations... 92 7.7 General conclusion ... 94

Samenvatting ... 95

Dankwoord ... 97

Curriculum Vitae ... 99

References ... 100

Summary

Skin layer mechanics

The human skin is composed of several layers, each with an unique structure and function. Knowledge about the mechanical behavior of these skin layers is important for clinical and cosmetic research, such as the development of personal care products and the understanding of skin diseases. Until today, most research was performed in vivo and focused on the mid-layer, the dermis. However, clinical and cosmetic applications require more detailed knowledge about the skin layers at the skin surface, the viable epidermis and stratum corneum, and the deeper lying hypodermis. Studying these layers in an in vivo set up is very challenging. The different length scales, ranging from μm for the stratum corneum to cm for the hypodermis, the interwoven layered structure and the inverse relation between penetration depth and resolution of non-invasive measurement techniques form major problems. As a consequence, hardly any data are available for the viable epidermis and hypodermis and reported data for stratum corneum are inconsistent. The aim of this thesis was therefore to characterize the mechanical behavior of individual skin layers in vitro and, for that, to develop the required experimental procedures. It was considered essential to perform experiments with samples of consistent quality in an accurate measurement set-up in a well-controlled environment. Various isolation and preservation methods were investigated on tissue performance, reproducibility and ease of handling.

Because of the inhomogeneous layered structure of the upper skin layers, mechanical properties of the stratum corneum and viable epidermis were determined for various loading directions. First, the stratum corneum and epidermis were subjected to shear over a wide frequency range and with varying temperature and humidity. The typical geometry of the upper skin layers required preliminary testing series in order to define the right experimental conditions to ensure reliable results. Subsequently, micro-indentation experiments were applied using a spherical tip with a relatively large

diameter. The Young‟s moduli were derived via an analytical and numerical method. Because of the complexity of measuring those skin layers, it was decided to focus on small deformations first.

For both types of loading, result were highly reproducible. The shear tests demonstrated that the shear modulus is influenced by humidity but not by temperature in the measured range. If the skin is compressed with an indenter, the stiffness of the epidermis and stratum corneum, which is about 1-2 MPa, is about a factor 100 higher than for shear. No significant differences in stiffness between the stratum corneum and viable epidermis were observed per loading type. The results of these tests prove that it is essential to take into account the highly anisotropy of the tissue in numerical models.

Rheological methods were developed to study the mechanical response of the subcutaneous adipose tissue. In the small linear viscoelastic strain regime, the shear modulus showed a frequency- and temperature-dependent behavior and is about 7.5 kPa at 10 rad/s and 37°C. Time-Temperature Superposition is applicable through shifting the shear modulus horizontally. A power-law function model was able to describe the frequency dependent behavior at constant temperature as well as the measured stress relaxation behavior.

Prolonged loading at small strains results into a dramatic stiffening of the material. Loading-unloading cycles showed that this behavior is reversible. In addition, various large strain history sequences showed that stress-strain responses are reproducible up to 0.15 strain. When the strain further increases, the stress is decreasing for subsequent loading cycles and, above 0.3 strain, the stress response becomes stationary. These results showing time and strain effects indicate that adipose tissue likely behaves as an (anti-)thixotropic material, meaning that a constitutive model should contain parameters to describe the build-up and breakdown of the material structure. However, further experimental research is needed to fully understand the thixotropic behavior before such a model can be worked out in detail.

In conclusion, this thesis evaluates the mechanical behavior of stratum corneum, epidermis and hypodermis using various in vitro set-ups. It was proven that for all skin layers reproducible results can be obtained. The research was aimed at developing reliable methods to determine the mechanical behavior of individual human skin layers. Future work should be focused on the relationship between mechanical properties and tissue deformation using imaging techniques and heading to the determination of the skin‟s failure behavior in relation to clinical and cosmetic treatments.

Chapter 1

1.1 Introduction

The largest organ of the human body, the skin, has a major role in providing a barrier against the hostile external environment. The skin prevents excessive water loss from the aqueous interior, the ingress of foreign chemicals and micro-organisms and provides strength and stiffness to resist mechanical loading. Other functions include insulation, temperature regulation and sensation. To fulfill these functions, mechanical stability is as important as mechanical flexibility. However, the mechanical balance of skin can be threatened by diseases, trauma, medical or cosmetic treatments. In order to understand the skin behavior following the onset of these conditions, knowledge of the mechanical behavior of healthy skin in normal conditions is essential.

Human skin is composed of several layers, each with a unique structure and function, but most research on its mechanical properties have ignored this non-uniform layered structure. For many clinical and cosmetic applications, however, knowledge of the mechanical behavior of the various skin layers is indispensible (Figure 1.1). For example, the benefit of transdermal drug delivery is that the microneedles exclusively damage the pain-free outer skin layer, the epidermis. Its mechanical response is therefore of particular interest. For needle insertion into the underlying dermal layer or for diseases such as pressure ulcers, the combined mechanical response of all individual skin layers is important. Although often not recognized, this is also the case during the removal of skin adhesives or the use of consumer products such as shavers. For all these applications, the subcutaneous fat layer contributes by attenuating or dispersing the external pressures, even when those are very small [1]. In addition, mechanical properties of the distinct skin layers are needed to grow them artificially, serving a wide application field. These include the development of artificial outer skin to substitute animal and clinical testing in evaluating drugs, cosmetics and other consumer products, and engineered fatty tissue facilitates large volume soft tissue augmentation in plastic surgery. Furthermore, the mechanical behavior of subcutaneous fat is critical for many other clinical treatments beyond the scope of this thesis, such as liposuction surgery and cellulite treatments.

To date, research on skin mechanics has mainly focused on full-thickness skin, the mid-layer (dermis) and the top mid-layer of the epidermis, the stratum corneum. The significance of a proper understanding of the mechanical behavior of the other part of the epidermis, the viable epidermis, and the subcutaneous fat tissue is not yet commonly felt. Indeed very limited experimental data is available for those layers. In addition, there is no consistency in data for the stratum corneum. Accordingly, the mechanical behavior of individual skin layers could not have been yet incorporated in numerical models. This thesis therefore focuses on the mechanical characterization of stratum corneum, epidermis and the subcutaneous adipose tissue. Before the scope and outline of the thesis is given, the anatomy of the skin and skin layer mechanics is shortly discussed.

(a) (b)

(c) (d)

(e) (f)

Figure 1.1 Clinical and cosmetic applications where the mechanical properties of separate skin layers are important: (a) transdermal drug delivery; (b) skin-device contact such as during shaving; (c) removal of adhesives such as ECG electrodes; (d) decubitus; (e) needle insertion procedures; (f) tissue engineering.

1.2 A mechanical view of skin anatomy and physiology

Mechanical properties of skin vary considerably and depend on body site, age, race and gender. Individual factors like exposure to UV irradiation, the use of creams and individual health and nutritional status can also affect the mechanical properties.

From the skin surface inwards, skin is composed of epidermis, dermis and hypodermis (Figure 1.2 ). The epidermis is mainly composed of cells migrating to the skin surface. The stratum corneum is considered as a separate layer because of its specific barrier properties. It consists of non-viable cells and is considered to be very stiff but pliable and wrinkled. The other part of the epidermis, the viable epidermis, is also wrinkled. The underlying layer, the dermis, is largely composed of a very dense fiber network dominating the mechanical behavior of the total skin. The deepest skin layer, the hypodermis or subcutaneous adipose tissue, is composed of loose fatty connective tissue. All skin layers contain microstructures like blood vessels, lymph vessels, nerve endings, sweat glands and hair follicles. The influence of these structures on the mechanical properties can be considered to be minimal in comparison to the bulk mechanical behavior caused by the main components of the skin layer.

As this thesis focuses on the mechanical behavior of the other layers, i.e. stratum corneum, viable epidermis and hypodermis, the anatomy and physiology of these skin layers are of particular interest.

Figure 1.2 Schematic representation of the different skin layers.

1.2.1 Skin topography

The topography of the skin surface is formed by the association of furrows, follicular orifices and sweat pores, and slightly protruding corneocytes. On most body sites, the

main furrows, called primary lines, are 70-200 μm deep, and follow at least two directions. The follicular orifices are located at the junction of the furrows, whereas the sweat pores are mainly found in the plateaus or in more superficial furrows, called secondary lines, being 20-70 μm deep. The third type of furrows separate groups of corneocytes. The network of furrows varies with age and gender.

The main function of the furrows is considered to be mechanical. By (partially) smoothing out, the skin surface and the epidermis can extend without loading the cells. The deeper the furrows and the steeper their sides, the higher their physiological range of extension. The direction of the higher extensibility is perpendicular to the direction of the main furrows. As a consequence, the stratum corneum in vivo hardly experience elongation stresses, but only unfolding. The furrows cannot be ignored when methods are developed to mechanically characterize the stratum corneum and the epidermis.

1.2.2 Stratum corneum

The stratum corneum is composed of corneocytes, which are hexagonal flat cells without a nucleus, held together by lipids and desmosomes in what is commonly referred to as a brick-and-mortar structure (Figure 1.3). The diameter and thickness range from 25 to 45 μm and approximately 0.3-0.7 μm, respectively [2,3]. The stratum corneum consists of 15-25 [3,4] layers of corneocytes, resulting in a total layer thickness of about 10-25 μm [5]. The lipids are arranged in lamellar sheets, which consist of membrane-like bilayers of ceramides, cholesterol, and fatty acids together with small amounts of phospholipids and glucosylceramides. The intercellular spaces, i.e. the distance between neighboring corneocytes, are about 0.1-0.3 μm [6]. Desmosomes, also called corneosomes, are specialized inter-corneocyte linkages formed by proteins and, together with the lipids, they maintain the integrity of the stratum corneum [7]. The lipids form the major permeability barrier to the loss of water from the underlying epidermis.

The stratum corneum, and viable epidermis, is continuously renewed within 6 to 30 days [8]. Cells are shed from the outside and replaced by new ones. Changes in structure, composition and function of the corneocytes occur as they move toward the outer skin surface. Cells of the deeper layers of the stratum corneum are thicker and have more densely packed arrays of keratins, a more fragile cornified cell envelope and a greater variety of modifications for cell attachment. Consequently, the deeper part of the stratum corneum has a major influence on its overall mechanical behavior. The outer stratum corneum cells have less capacity to bind water. The cells in the outermost stratum corneum have a rigid cornified envelope and in the same area, the desmosomes undergo proteolytic degradation.

Although the corneocytes are non-viable, the stratum corneum is considered to be fully functional, particularly in terms of barrier properties, and retains metabolic functions [9].

(a) (b)

Figure 1.3: Morphology of the stratum corneum. (a) schematic drawing (b) cryostat section of normal human skin treated with Sorensen’s alkaline buffer and methylene blue. Obtained from Marks [10].

The mechanical properties of both stratum corneum and viable epidermis are influenced by environmental conditions such as relative humidity (RH) and temperature. In addition, topical applications of either pure water, moisturizers or emollients alters the hydration state of the stratum corneum, significantly modifying some of its mechanical properties. Under normal conditions, the hydration in the stratum corneum conditions varies from 5-10% near the surface up to 30% near to the transition with the viable epidermis. Bound water associated with proteins and lipids accounts for 20-30% of the total water volume. The total water content varies little between 30% and 60% RH, although it increases considerably at higher values [11]. When fully hydrated, the stratum corneum swells to twice its normal thickness. In an in vitro situation, however, the stratum corneum can increase up to 400% of its original thickness [12]. This highlights the constraints imposed on the stratum corneum in vivo.

1.2.3 Viable epidermis

The viable epidermis is a layered structure, consisting of three layers or „strata‟. The bulk of epidermal cells are the keratinocytes, which migrate upwards to the skin surface where they become non-viable. Other cell types within the viable epidermis include melanocytes, Langerhans cells and Merkel cells.

Keratinocytes change their shape, size and physical properties when migrating to the skin surface. Indeed the morhology of an individual keratinocyte correlates with its position within the epidermis and its state of differentiation, which is reflected by the different strata: the stratum basale, the stratum spinosum and the stratum granulosum (Figure 1.4). The deepest layer is the stratum basale in which cell division occurs. It consists of 1 to 3 layers of small cubic cells. In the next layer, the stratum spinosum, the cells are larger and polyhedral in nature and are connected by desmosomes, which are symmetrical laminated structures. The keratinocytes adopt a more flattened morphology at higher layers of the stratum spinosum. In this layer, they are associated with lamellar granules, which are lipid-synthesizing organelles that migrate toward the periphery of the cell and eventually become extruded into the intercellular compartment in the next layer, the stratum granulosum. At this stage of differentiation, the degradation of

mitochondria and nuclei is apparent and the cytoplasm of the flattened cells become increasingly filled with keratohyalin masses and filaments. Furthermore, the cell membrane becomes gradually thicker.

The thickness of the viable epidermis varies roughly between 30-100 μm [13], accomodating between 5 to 10 cell layers. The cells are communicating by very strong desmosomes in the very compact tissue; the intercellular spaces occupy less than 2% of the volume [5,14]. Therefore, the mechanical integrity of the viable epidermis is considered to be stronger than other soft tissues.

Because of its non-vascular structure, the epidermal cells are nourished from plasma that originates in the dermal blood vessels such that the nutrients transport across the epidermal-dermal junction.

Figure 1.4: Morphology of the epidermis. In the schematic drawing the nucleus (N), the keratin filaments (KF), the desmosomes (D) and the lamellar granules (LG) are depicted. The histological section is taken from the skin of a young woman, obtained from Montagna et al. [15].

1.2.4 Dermal-epidermal junction

The boundary between the dermis and epidermis is called the dermal-epidermal junction, which provides a physical barrier for cells and large molecules. Four distinctive zones in this strong junction can be identified: 1) the plasma membrane and hemidesmosomes of the basal keratinocytes adhered to the junction, 2) the lamina lucida zone with anchoring filaments, 3) the lamina densa, and 4) the amorphous sublamina densa fibrillar zone (see Figure 1.5). The degree of attachment is enhanced by parts of the epidermis penetrating the papillary dermis resulting in large cones, called rete ridges or papillae [16]. The major point of weakness is considered to be the lamina lucida [17]. The dermal-epidermal junction length over a straight line ranges from 1.1 to 1.3 units [5].

stratum corneum basal layer granulous layer spinous layer N D KF LG

Figure 1.5: Ultrastructure of the dermal-epidermal junction.

1.2.5 Dermis

The dermis can be divided into two anatomical regions: the papillary and reticular dermis. The papillary dermis is the thinner outermost portion of the dermis, constituting approximately 10% of the 1-4 mm thick dermis. It contains relatively small and loose distribution of elastic and collagen fibrils within a significant amount of ground substance. Its content in water and vascular volume show physiological variations that can alter the mechanical behavior of skin as a whole. In addition, collagen and elastin fibers are mostly vertically oriented in the papillary region and connect to the dermal-epidermal junction. In the reticular dermis, fibers are horizontally oriented.

The dermis has a mainly mechanical function. The reticular dermis is able to extend up to about 25% by stretching the collagen fibers, whereas it can be squeezed due to the capacity to displace the ground substance laterally. The elastic fiber network ensures full recovery of tissue shape and architecture after deformation. The amorphous ground substance acts as a viscous gel-like material, which does not leak out of the dermis, even under high pressure. The permanent tension in the reticular dermis generates the folding of the overlying structures and hence, the skin surface. The fiber network in the papillary dermis contributes to the protection of vessels and cells against mechanical insults. In the papillary dermis, the microvasculature consists of papillary loops exchanging with extravascular elements and a horizontal plexus in which the loops emerge. Although the vascularization throughout the dermis appears relatively sparse, the supply of the papillary loops is ensured by arterioles irrigated from the deep dermis.

1.2.6 Hypodermis

The hypodermis is defined as the adipose tissue layer found between the dermis and the aponeurosis and fasciae of the muscles. Its thickness varies with anatomical site, age, sex, race, endocrine and nutritional status of the individual. The subcutaneous adipose tissue is structurally and functionally well integrated with the dermis through nerve and vascular networks and the continuity of epidermal appendages, such as hairs and nerve endings.

The bulk of subcutaneous adipose tissue is a loose association of lipid-filled cells, the white adipocytes, which are held in a framework of collagen fibers. However, only one third of adipose tissue contains mature adipocytes [18], with the remainder being stromal-vascular cells including fibroblasts, leukocytes, macrophages, and pre-adipocytes [19]. Adipose tissue has little extracellular matrix compared to other connective tissues.

Stored fat is the predominant component of the adipocytes; where the lipid droplet can exceed 50 μm. The cytoplasm and nucleus appears as a thin rim at the periphery of the cell (Figure 1.6). The diameter of the entire white adipocyte is variable, ranging between 30 and 70 μm [18]. Collections of white adipocytes comprise fat lobules, each of which is supplied by an arteriole and surrounded by connective tissue septae. Each adipocyte is in contact with at least one capillary, which provides the exchange of metabolites and allows the adipocytes to function effectively. It is interesting to note that the subcutaneous adipose tissue of the lower trunk and the gluteal thigh region has a thin fascial plane dividing it into superficial and deep portions. Morphological differences are observed between these two adipose tissue layers [20].

(A) (B)

Figure 1.6: Schematic drawing (a) and histological section (b) of hypodermis, or subcutaneous adipose tissue, showing white adipocytes (WA) with the nucleus (N) at the periphery. The adipocytes are in contact with the blood circulation via arterioles which branches the larger arteries (A) and veins (V).

A V

N

The mechanical functions of the subcutaneous adipose tissue include allowing the overlying skin to move as a whole, both horizontally and vertically, and the attenuation and dispersion of externally applied pressure.

1.3 Review of skin layer mechanics

Measurement methods and mechanical properties of skin have been extensively reviewed in the literature [5,21,22]. Therefore, given the focus of the present work, focus will be limited to studies on the behavior of stratum corneum, viable epidermis and hypodermis. More specifically, they include force-elongation data, either in vivo or in vitro, and currently available constitutive models.

1.3.1 In vivo vs in vitro experiments

When measurements on skin mechanics are performed in vivo, the human skin exists in its natural pre-stress and skin relief. The number of in vivo measurement methods is, however, limited [22] and a numerical-experimental approach is usually adopted. In any in vivo study, it is difficult to determine the contribution of each individual skin layer to the overall skin response, whereas in vitro measurement methods offer the potential to perform well-controlled experiments on individual skin layers. Another benefit of the latter is that all forms of mechanical testing can be applied and a wide range of reliable direct measurement methods becomes available. However, due to the limited availability of skin grafts, the number of experiments, the variety of skin types, and the variety of body sites can be problematic.

The appropriateness of in vitro experiments on the stratum corneum should be carefully considered. In vivo, the stratum corneum partly unfolds when the total skin is stretched, but does not elongate. Full extension of the stratum corneum occurs in critical, extra-physiological situations due to disease, trauma, clinical or cosmetic applications.

1.3.2 Mechanical behavior of the stratum corneum

Force-elongation curves at constant elongation rate demonstrate one, two or three phases depending on the hydration level in the in vitro experiment (Figure 1.7) [23]. The first phase, up to a 10% extension, is considered to represent purely elastic behavior. The next phase, absent at low RH, is an irreversible elongation with a low slope, with strains ranging from 20-125%. In addition, fully hydrated stratum corneum exhibiting a final phase, where strain hardening is observed before rupture, at approximately 200% extension. The slope becomes steeper at increasing elongation rates, as would be predicted of a viscoelastic response. Although the corneocytes are very elongated in tensile testing, the final rupture is always extracellular and most likely at the desmosomes [8].

From the 1970s, various authors have reported tensile testing [8,23-27]. Subsequently, torsional techniques were developed to measure the stratum corneum behavior in vivo [28-31]. More recently, indentation techniques were introduced to determine the Young‟s modulus in vitro [32,33], and also in vivo indentation tests have been

performed [34]. Furthermore, imaging techniques such as ultrasound and magnetic resonance elastography have been used to estimate mechanical properties [22,33].

Reported Young‟s moduli vary considerably encompassing values from a few MPa to GPa [24,25,35,36]. For example, the estimated tensile moduli for various RH is shown in Figure 1.8. As indicated in this figure, the stiffness of the stratum corneum varies from rubber-like at high RH to nylon-like at low RH values. The differences may be due to a combination of reasons, such as regional differences, anisotropy, differences between species, but also test conditions, such as sample preparation and difficulties in controlling sample dimensions and environmental conditions. A general trend, however, is a more pronounced decrease of the elastic modulus beyond 60% RH. At a constant RH, the stratum corneum hydration increases by 50% when the temperature rises from 20°C to 30°C. The influence of temperature decreases to a minimum beyond 90% RH. More common trends due to an increase in RH or temperature include an increase of the maximum extension and work of rupture, and a reduction of the force at rupture [23,25,37]. Furthermore, stratum corneum behaves isotropically in transversal plane only [36].

Preconditioning effects have not been reported for stratum corneum, which represents an important difference with the whole skin. This finding indicates the absence of mobile components in the stratum corneum [36].

Current constitutive models of the stratum corneum are based on traction, relaxation and creep tests [5]. From the experimental tests, it is important that the model accomodate elasticity, non-linear viscosity and strain hardening parameters. However, the association

Figure 1.7 Typical force elongation curves for the stratum corneum at different RH showing different phases: the elastic phase (I), the plastic phase (II) and the strain hardening phase (III). Obtained from [23].

10 20 30 40 I II III 98% RH 76% RH 30 120 32% RH Elongation [%] L oa d [g ] in v ivo ra n g e

between the defined parameters and the anatomical components has yet to be determined.

Figure 1.8 An overview of Young’s moduli of stratum corneum as function of the RH derived from in vitro tensile tests.

1.3.3 Mechanical behavior of the viable epidermis

Only recently, a few studies have focused on the viable epidermis. From an indentation approach, a local Young‟s modulus of a few MPa has been reported for the viable epidermis of murine ear skin [38,39]. However, it is recognised that murine skin exhibits a higher density of hair follicles and a very thin epidermis compared with human skin. Indeed a combined experimental-numerical approach on in vivo human skin yielded an estimated Young‟s modulus of about 0.5 kPa for the upper human skin layers including the papillar dermis [1,39]. The authors hypothesized that this low value was due to the negligble influence of the stratum corneum on the overall mechanical response of the skin, when suction was performed with small aperture sizes. Due to the dearth of experimental data, a constitutive model describing the mechanical behavior of viable epidermis is not yet available.

1.3.4 Hypodermis

A limited number of studies is available regarding the mechanical behavior of subcutaneous adipose tissue subjected to applying shear [40], compression [40,41], indentation [42,43] or suction [1,39-41,44]. Young‟s moduli varied from a few kPa to values in excess of 100 kPa.

All studies provide limited descriptions of the overall mechanical behavior as they were developed for very specific applications. Consequently, an appropriate constitutive model based on experimental data is not available yet. Indeed current models are either limited to small strain behavior [39,45] or based on other soft connective tissues.

1.4 Aim and Outline

The objective of this thesis is to develop appropriate experimental techniques and procedures, which will enable the characterization of the mechanical behavior of individual skin layers in vitro. The focus is on those skin layers for which available data is relatively scarce, i.e. the viable epidermis and hypodermis, and/or inconsistent as in the case for the stratum corneum. The results should provide insight into the relationship between the mechanical responses to the structure of the various skin layers and, hence, provide better understanding of the way a treatment or disease affects the skin behavior. Furthermore, the experimental data should provide suitable input for constitutive models. Previous studies, such as the various in vitro tensile tests on the stratum corneum, have indicated that differences in mechanical properties of the epidermis and stratum corneum are not solely caused by variations in humidity and temperature, but are influenced test conditions, anisotropy, sample preparation, etc. It is therefore essential to perform experiments with samples of consistent quality in an accurate measurement system in a well-controlled environment. This will be initially achieved in relatively simple small strain experiments in various directions under different environmental conditions. If this small strain behavior is reproducible and well-understood, then it is appropriate to extend the work to examine the non-linear behavior.

In order to obtain in vitro samples of consistent quality, various isolation and preservation treatments are first thoroughly investigated for both skin layers (Chapter 2). Subsequently, a rheological measurement system has been designed to measure the shear response of thin, soft tissues in a controlled environment (Chapter 3). A micro-indentation method has been adapted to enable the measurement of loading perpendicular to the skin surface (Chapter 4). Because viable epidermis cannot be isolated as a single layer, a numerical model is introduced to predict its behavior from the experiments on stratum corneum and whole epidermis.

Subsequently, rheological methods are developed to study the linear shear response of subcutaneous adipose tissue (Chapter 5). From those results, a constitutive model describing the linear viscoelastic behavior of subcutaneous adipose tissue at small strains has been developed. Then, a set of experiments were designed to study both the large deformation and time-dependent behavior (Chapter 6).

Finally (Chapter 7), a general discussion evaluates the selected measurement methods for the skin layers and these outcomes, as well as the significance of the findings of this work for various applications.

Chapter 2

Isolation and preservation methods for

the epidermis and stratum corneum

The contents of this chapter are based on M. Geerligs, D. Bronneberg, P.A.J. Ackermans, C.W.J. Oomens, and D.L. Bader, Isolation and preservation methods for the

2.1 Introduction

Ex vivo human skin grafts provide a cost-effective alternative to animal and clinical testing. Various industries, such as the cosmetic, household product and pharmaceutical, could benefit from in vitro studies to evaluate drugs and a range of consumer products. Skin models are already used in many transdermal drug delivery and percutaneous absorption studies, as well as in irritancy and toxicology studies. Studies on ex vivo skin increase the fundamental knowledge on both structural and mechanical properties of skin. In addition, studies on isolated skin layers, such as the epidermis or stratum corneum, could provide an insight into the specific contribution of each layer to the overall skin response. Skin models enable improved control of experimental conditions, i.e. temperature, hydration level, and offer the potential to perform well-controlled in vitro experiments. In order to obtain meaningful results, it is of utmost importance that the structural integrity and viability of the skin are maintained.

The epidermis, the outermost skin layer, is directly contiguous to the external environment and acts as a permeable barrier. It prevents excess water loss from the aqueous interior and protects the internal tissue against mechanical insults, UV irradiation and the ingress of foreign chemicals and micro-organisms. Due to the extraordinary nature of the epidermis, its complete isolation while maintaining its structural integrity remains a challenge. The keratinocytes are surrounded by a poor extracellular matrix and lack the support of a fiber structure, which provides the strength and stiffness of most biological tissues. Within the epidermis, the mechanical properties are determined by the rigid tonofilament cytoskeleton and the numerous desmosomes to which the filaments are anchored at the periphery of the keratinocytes. At the epidermal-dermal junction hemidesmosomes anchor the epidermis to the dermis (see Figure 1.5). These hemidesmosomes or the adjacent anchoring filaments need to be disrupted to fully separate the epidermis from the dermis.

In order to maintain the complex structure of the stratum corneum during isolation, it is important to preserve the curvature. The architecture of the stratum corneum is widely established as a solid brick-and-mortar structure, with flat corneocytes surrounded by a matrix of lipid enriched membranes strongly held together by desmosomes.

Due to the high number of plastic and cosmetic surgery procedures, such as abdominoplasty and breast reduction, there is an increased availability of ex vivo human skin. Whether a skin graft can be successfully used as skin model during in vitro experiments depends on the nature of the tissue. The integrity of the skin tissue mainly depends on the age of the subject, as well as on the donor body site. Furthermore, within one skin graft, its structure might change as a result of disease or prior treatment. These factors are usually reflected in tissue changes, such as convolutions of the epidermal-dermal junction, thickness of epiepidermal-dermal strata, cell shape and surface folding, but may also lead to qualitative and quantitative differences in the various epidermal components

[46]. To obtain the best experimental outcome from in vitro studies, it is important to use structurally and functionally intact models.

In order to use the available intact skin grafts with optimal efficiency, factors such as cleaning, preservation, and storage should be adequately addressed. In various studies, such as transdermal drug delivery, percutaneous absorption studies, irritancy and toxicology studies, an intact skin barrier is essential. Furthermore, adequate preservation is crucial for maintaining the viability and integrity of the skin tissue. Tissue damage such as the creation of vacuoles are easily induced and the selection of a proper tissue storage method is therefore important.

Evaluation techniques to assess skin viability during storage have been extensively described [47-49]. Common methods to assess viability include Trypan blue dye exclusion, tetrazolium reductase activity, oxygen consumption rates, lactate and glucose levels, and NMR spectroscopy. Structural integrity is usually assessed by histological routines or imaging techniques.

This paper aims to critically review various isolation methods for the epidermis and stratum corneum and preservation methods useful for in vitro research on split-thickness skin, epidermis and stratum corneum. Existing reviews are considered to be out of date and do not include recent work from the host laboratory [46,50-52]. No standards exist, thus inter-study comparisons are problematic. In addition, much of the existing data may have been influenced by the specific preparation technique, which have been employed. Accordingly, the present paper describes mechanical, ionic change, heat, enzymatic digestion and irradiation techniques for isolation of the skin layers. The advantages and disadvantages of each technique are discussed in terms of maintaining the skin integrity and ease of handling. In addition, the influence of various storage conditions on the skin structure and viability are discussed.

2.2 Skin preparation and analyses

General steps in the preparation of skin samples used in the present experiments are described below, as well as the analysis techniques used to study the skin structure and viability.

2.2.1 Skin preparation

Human skin was obtained from female patients undergoing abdominoplasty. The research proposal for our studies was approved by the Medical Ethics Committee of the Catharina Hospital, Eindhoven, the Netherlands. Immediately after excision, the skin is brought to the laboratory for further processing. Here, the skin is placed on a stainless steel plate covered with paper towels to absorb body fluids. The skin surface is cleaned with pure water. Using multiple forceps, the skin graft is stretched and fixed to the stainless steel plate (Figure 2.1a). Subsequently, split-thickness skin samples, varying in thickness from 100-400 µm, are produced using a commercial dermatome (D42, Humeca, The Netherlands) (Figure 2.1b).

(a) (b)

Figure 2.1: Skin is stretched using forceps (a) and dermatomed (b).

(b)

(a) (c)

Figure 2.2. (a) Full thickness skin stained with aldehyde-fuchsin to visualize the stratum corneum (SC), viable epidermis (VE), papillar dermis (PD) and reticular dermis (RD); (b) Dermatomed skin with a set thickness of 100 μm consists of the epidermal layer only; (c) In some cases, however, some papillar dermis is still attached.

2.2.2 Histological examination

In order to examine tissue structure, samples were fixated in 10% phosphate-buffered formalin and processed for conventional paraffin embedding. The sections were cut into 5 μm slices and stained with aldehyde-fuchsin and yellow green SF (Merckx) or standard heamotoxilyn and eosin (H&E) staining. The tissue morphology was studied by light microscopy. The aldehyde-fuchsin staining is used to clearly identify the different skin layers, namely the stratum corneum, viable epidermis, papillar dermis and reticular dermis (Figure 2.2a). The structural integrity is examined by using the H&E staining.

SC

VE

PD

RD

SC

VE

SC

VE

PD

2.2.3 Analyses of skin viability

Skin viability was studied by using the colorimetric MTT (Thiazolyl Blue Tetrazolium

Bromide) assay. Skin samples with a diameter of 8 mm were placed in a 24 wells-plate

containing 300 µl of 1 mg/ml MTT solution in PBS in a well (Phosphate Buffered Saline). The plates were incubated at 37C and 5% CO2 for a period of 3 hours. After

incubation, the skin samples were removed and gently blotted with tissue paper, before completely submerging them in 2 ml 2-propanol per well. The extraction plates were placed in sealed bags to reduce evaporation and were gently shaken for 2 hours at room temperature to extract the reduced MTT. The absorption of the extractant was measured at 570 nm, using plain extractant as blank.

2.3 Epidermal isolation techniques

Isolation techniques for the epidermis can be divided into the following categories: mechanical, ionic change, heat, enzymatic digestion and irradiation techniques. The effectiveness of each is summarized in Table 2.1 at the end of the section in terms of actual cleavage plane, maintaining of both cell viability and tissue integrity.

2.3.1 Mechanical separation

Cutting by using a dermatome

Van Scott et al. [53] recommended a stretching method for separating the epidermis from the dermis. The method involves manually stretching the skin to its limit over a slightly convex wooden surface, and anchoring it in place by means of thumbtacks. A razor blade or scalpel is used to scrape off the epidermis. Subsequently, the epidermis is grasped by tweezers to gently detach a continuous sheet. However, damage can be easily induced in the epidermis using this relatively crude stretching technique. The severity of this damage depends on the vigour of scraping and the degree of stretching. The development of keratomes, either handheld devices or as part of a mechanical device, has improved the reproducibility of this stretching technique.

In the present study, a cordless, battery operated dermatome was used. As previously mentioned, ex vivo skin was mounted on a stainless steel plate to facilitate the cutting process. When the dermatome was set to 100 μm, samples of the epidermis could be obtained. In some cases, however, some papillar dermis was still attached to the epidermal specimens (Figure 2.2). Due to the presence of rete ridges, it was highly unlikely that the cutting plane went through the dermal-epidermal junction only. However, the number of skin layers present in the separated tissue can be assessed visually; with the yellowish translucent epidermis being easily distinguishable from the white opaque dermis. A MTT-test demonstrated that the dermatomed skin retained its viability for 100%, which is in agreement with Wester et al. [54].

The defined geometric shape of the specimen is very convenient for assessing its mechanical properties. It is assumed that the mechanical properties of the present papillary dermis are similar to the surrounding epidermal tissue, because no differences

in shear properties were found between 100 and 200 μm thick split-skin samples (see Chapter 3).

Suction device

Suction blisters can be produced by applying suction cups on the skin, in both in vivo and in vitro experiments. In vivo separation of the human epidermis was first reported in 1964 [55]. Kiistala et al.(1968) found that a blister could be induced within 130 minutes with a suction gap of 25 mm. The diameter of a suction cup may vary from 15-50 mm depending on body site. To avoid tissue damage, the pressure within the cup had to be maintained at 200 mm Hg or above. The cleavage occurs in the plane through the lamina lucida, leaving the lamina densa on the dermis and retaining an intact, viable basal cell layer. However, enlargement of intercellular spaces due to considerable stretching might cause large vacuoles in keratinocytic cytoplasm [50,56].

Suction blister time depends on factors such as suction pressure, individual variation and regional differences as well as temperature, but does not depend on cup size. Because of the low reproducibility caused by individual variations that cannot be controlled, this method is considered to be unfavourable.

2.3.2 Ionic change

An earlier method to isolate the epidermis involved its maceration in dilute acetic acid. Cowdry [57] described that dilute acetic acid causes swelling of collagen fibers which decreases their cohesive strength and, therefore, the binding of epidermis to dermis. In addition, it was found that collagen fibers also swell in an alkaline environment. These methods, however, are toxic to epidermal cells and are therefore no longer used [58]. In addition, EDTA (ethylenediamine tetraacetic acid) has been used to obtain epidermal sheets [59]. The location of the split changes according to the duration of the treatment. For example, after 30 min incubation in 0.01 M EDTA at pH 7.4 the split occurred in the lower granular layer, whereas after 45 min it was in a spinous-suprabasilar location and after 60 min or more it occurred at the dermal–epidermal junction. In adition, intracellular oedema increases with time. Accordingly, this is not considered to be a favourable method for epidermal separation.

After prolonged incubation in 1 M NaCl at 4°C, the epidermis can also be easily removed from the dermis with forceps. The split occurs through the lamina lucida. Nevertheless, mitochondrial swelling within the keratinocytes was noted [50]. Although no other degenerative features have been reported, epidermal components may have been diminished or modified during the long incubation times of 24 to 96 hours [60].

Prolonged incubation in PBS is also known to separate the epidermis from the dermis. Indeed after 72-96 hours at 37°C, the epidermis can be readily peeled off [61]. In contrast to the above techniques, where the split occurs through the lamina lucida, the split is closer to the epidermal site of the dermal-epidermal junction [61].

Since no intact viable epidermal sheets can be obtained using any of the techniques based on ionic change, they are not considered suitable for epidermal isolation.

2.3.3 Heat

Separating the epidermis from the dermis using a hot plate is a simple and rapid method [58]. It was reported that the skin is heated up to 50 to 60C for 30 s. To maintain enzyme activity, mild heat treatment at 52C for 30 s is required. Separation occurs at the basal cell layer. Depending on the exact conditions, release of enzymes, cytolysis and cell separation may occur. However, it has been claimed that heat does not modify fibrous proteins within isolated epidermis [62]. Although heating can easily cause tissue dehydration, this can be minimized by increasing the humidity of the environment or by placing the skin in a sealed bag in hot water, instead of using a hot plate. After heating, the epidermis can be gently peeled from the dermis.

In the present studies, human skin samples were heated on either a hot plate and in a sealed bag. The former process appeared to flatten the undulating epidermal structure, while the papillae remained intact after heating in a sealed bag in hot water. Much longer heating times were needed than mentioned in literature. The epidermis could be peeled from the dermis after more than 5 minutes.

For both heat separation techniques, structural tissue damage occured as evidenced by the presence of vacuoles and a disrupted basal layer (Figure 2.3). It has been previously reported that heat treated skin (60°C for 1 minute) and heat-separated epidermis and dermis significantly lose viability [63]. Furthermore, some practical problems arose when using a hot plate, such as curling of the dermal tissue and uneven separation of the epidermis over the complete skin surface due to gradual thermal diffusion.

(a) (b)

Figure 2.3. Histological sections of epidermis isolated using heat by means of a hot plate (a) or placing the epidermis in a sealed bag in hot water (b). A standard H&E staining has been used.

2.3.4 Enzymatic digestion

Trypsin

Epidermal separation by means of trypsin has been widely used, although some conflicting results have been published. For example, Briggeman et al. [64] reported that the epidermis is isolated by the cleaving effect of trypsin, whereas other authors reported that many basal cells remain loosly attached to the basement membrane after trypsin treatment [65,66]. The epidermis can be easily peeled from the dermis using 0.1-0.3%

trypsin in a saline solution supplemented with calcium and magnesium at 4°C. However, these conditions also induce a high level intra-epidermal split at the spinous-granular interface [46]. Inconsistencies within the reported findings seem to be related to various factors such as size and thickness of the skin sample, enzymatic concentration and its solvent, incubation time and temperature. In addition some side-effects are noted following trypsin treatment such that recovery may take up to a few days [46]. All these factors lead to inconsistent epidermal separation following treatment.

Thermolysin

The epidermis can easily be separated from the dermis following incubation at 4C for 1 h in a solution containing 250-500 g/ml thermolysin, a proteolytic enzyme more generally used for protein analysis [65]. Thermolysin can be dissolved in sterile magnesium free PBS containing 1 mM CaCl₂ at pH 7.8. However, to ensure complete penetration of the enzyme, it is advisable to remove the subcutaneous fat and the lower dermis from the specimen. Light and electron microscopy revealed that the separation occurred at the lamina lucida and that the hemidesmosomes were selectively disrupted [65]. By contrast, Willsteed et al.[50] noticed an intraepidermal split, without any lamina lucida separation.

Dispase

Dispase II (Roche Diagnostics) has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis [67,68]. This proteolytic enzyme is able to cleave the basement membrane zone region while preserving the viability of the epithelial cells.

Based on recommendations from the supplier, 2.4 U/ml dispase in 50 mM HEPES/KOH buffer pH 7.4 with 150 mM NaCl was used in the present studies to separate the epidermis from the dermis. Fresh skin samples of various sizes were placed on top of sterile gauzes in 6 cm diameter petri dishes containing 5 ml of 2.4 U/ml Dispase II. The stratum corneum of the skin samples was not exposed to the enzymatic solution during the separation process to minimize loss of the skin barrier integrity. After overnight incubation at 4C and thereafter 10 min at 37C, the epidermis was gently peeled from the dermis using tweezers. In agreement with literature, the present study demonstrated that the bottom surface of the separated epidermal sheet retained its rete-ridges and hair follicles with sebaceous glands and the eccrine sweat glands retained their undistorted shape [68] (Figure 2.4). The cleavage occurred in the lamina densa.

This isolation method is very suitable for generating intact epidermal sheets. The best results were obtained when split-thickness skin samples of roughly 300 µm, which were then enough to facilitate enzyme diffusion. Therefore, it is recommended to dermatome skin grafts prior to performing the enzyme treatment.

Figure 2.4. H&E staining of epidermis separated with Dispase.

2.3.5 Microwave irradiation

Sanchez et al. [69] explored the effects of microwave irradiation on epidermal-dermal separation. Epidermal samples were obtained after incubation in 0.02 M EDTA in PBS and microwave irradiation with 4 pulses of 420 watts for 5 sec, with a total incubation period of 4 min. The hemidesmosomal junctions are then disrupted, whereas an additional incubation time may affect keratinocyte junctions. Microwave irradiation has been widely used for tissue fixation and immunostaining.

Care should be taken to avoid damage to the tissue integrity. It is reported to be essential to use the prescribed buffer and specifically adhere to the recommended microwave exposure times. Nevertheless, microwave irradiation seems to be a rapid method for separation of the epidermis from the dermis.

Table 2.1: Critical of isolation techniques used for epidermal tissues. Techniques that are highlighted, are investigated in our laboratories.

2.4 Isolation techniques for the stratum corneum

Isolation techniques for the stratum corneum can be divided into the following categories: mechanical, chemical and enzymatic digestion techniques. The effectiveness

Type Method

Treatment

duration Cleavage plane

Tissue integrity

Tissue

viability Reproducibility

Mechanical Dermatome < 1 hr variable + + +

Suction < 2hrs lamina lucida 0 0

-Heat 5 min basal layer - - 0

Ionic _NaCl 24-96 hrs lamina lucida 0 n.a.* 0

change EDTA > 1 hr n.a. - n.a.*

-PBS 72-96 hrs hemidesmosomes - 0 0

Enzymatic Trypsin 1-24 hr variable - 0

-digestion _{Thermolysin 1 hr hemidesmosomes + + n.a.*}

Dispase 24 hrs lamina densa + + +

Irradiation Microwave 5 min hemidesmosomes 0 n.a.* +

of each technique is summarized in Table 2.2, in terms of maintaining both cell viability and tissue integrity.

2.4.1 Mechanical separation

Stratum corneum separating by cutting techniques is complicated due to the inherent curvature of the skin. However, the thickness of the stratum corneum has little variation, such that flattening of the skin might improve mechanical separation. It has already been shown that the skin relief dramatically decreases when a microscope slide is placed on top of it [70]. In the present study, topography measurements were performed on unloaded and loaded skin with a PRIMOS (GFM, Germany), using light profilometry to assess the surface roughness. A piece of skin of 20x20 mm was placed on a microscope slide after removal of the subcutaneous fat layer. First, the initial surface roughness parameters were measured. Then, another microscopic glass slide was placed on the upper surface of the specimen and pushed down with two weights of 100 g on each side. Again the roughness parameters were determined. Preliminary testing showed that the microscopic slide on top was not detected by the system and did not influence the measurement output. A significant decrease in skin surface roughness was measured, with a mean value of 42 μm in a loaded configuration compared with 85 μm in the unloaded state. The latter is comparable to what can be found in literature [5]. Nonetheless, the surface roughness in the loaded state was still at least three times the thickness of the stratum corneum.

Following the topography measurement, the sample was maintained between two plates and stored at -80°C. In order to retain the flattened state of the skin sample, the sample was cut using a cryotome. The surface of the stratum corneum was aligned with the cutting system to obtain the stratum corneum using a single cut with a thickness of 20 μm. The stratum corneum sheets have some other epidermal strata attached and cavities (Figure 2.5).

(a) (b)

Figure 2.5. Stratum corneum isolated from flattened skin. Due to the skin curvature, other epidermal strata and cavities are still present. Transversal sections of the obtained sheets are depicted with 5x (a) and 40x (b) enlargement.

2.4.2 Chemical separation

Cantharidin blister procedure

This method, however, has only been reported up to the early seventies [8,23]. Cantharidin was impregnated into 1 cm diameter disks of filter paper and placed under occlusive patches rather than applied directly to the skin surface in a volatile solvent. The disks were removed after 4 hours and protective caps were placed over the forming blisters to prevent damage to the samples. The blister tops were surgically excised and the loose underlying wet cells removed by gentle swabbing. Since the discovery that cantharidin is toxic, it is not permitted to use it for skin treatments anymore.

Ammonia vapour

In the sixties and seventies, it was common to isolate stratum corneum through exposure to ammonia vapour. The latest protocols reported around 30 min exposure to separate the dermis and epidermis [71,72]. Adherent wet cells are subsequently removed with a cotton swab such that the stratum corneum sheet remains [73]. Thereafter, the stratum corneum sheet was allowed to dry on silicone-coated paper at ambient conditions. In addition, it was noticed that the success of this treatment is variable. Since more consistent techniques causing less damage became available, this method is no longer used.

2.4.3 Enzymatic digestion

Trypsin

The working of trypsin throughout the epidermal strata has been extensively studied [73]. It appeared that the architecture of the stratum corneum remains unaffected by trypsinization. Corneodesmosomes and composite desmosomes shared by corneum and granular cells are normal. Tonofilaments attached to these junctions also appear unchanged [73]. However, concentrations of trypsin above 0.125% might damage the stratum corneum such that its elastic properties change [5].

In order to enable the working of trypsin on the epidermal cells, the subcutaneous fat layer and the lower dermis has to be removed. In our laboratories, the remaining skin was immersed in a porcine 0.1% trypsin (SV30037.01, Hyclone) solution in PBS (Phosphate Buffer Saline). For quick processing, the samples were then placed for over 2 hours in an incubator at 37°C. For this study, dermatomed skin of approximately 300 μm thick and a surface area of 2 cm2 was placed in 3 ml trypsin. Similar results can be obtained through an overnight culture at 4°C and 15 min at 37°C. Due to the lipids within the stratum corneum, the thin layer floats to the surface while the remaining epidermis sinks to the bottom. In order to prevent post trypsinization effects, stratum corneum is rinsed with distilled water a few times to wash out trypsin and treated with anti-trypsin. The overnight protocol can be considered as the golden standard, which is frequently described and commonly used within several research fields.

Figure 2.6. (a) After staying overnight at 4°C, the extracellular matrix of the viable epidermis is still attached to the stratum corneum; (b) Only stratum corneum is obtained after leaving the skin sample for 1 hour at 37°C.

Table 2.2: Overview of effectiveness of isolation techniques for the stratum corneum. Techniques that are highlighted, are investigated in our laboratories.

2.5 Preservation of the upper skin layers

This section discusses preservation techniques regarding in vitro skin research. It is assumed that these techniques are equally suitable for all skin grafts, i.e. full-thickness, split-thickness, and epidermal grafts. From studies on skin grafts used as burn wound dressings, it is known that in order to provide the best clinical outcome, skin grafts should be properly preserved. When procuring cadaver skin for banking, the cadaver donor should be cooled as soon as possible to avoid/minimize structural tissue changes, i.e. changes in basement membrane components [74], and to maintain viability. Within 12 to 30 hours from harvest, post-mortem skin allografts exhibit an average viability index of 75% with little variation, which decreases to 40% within 60 hours. In addition, Bravo et al. [54] found that human cadaver skin grafts only exhibited approximately 60% of the metabolic activity found in fresh skin samples from living surgical donors. However, the availability of skin grafts from living donors is limited to certain body sites.

Currently available methods used by skin banks for storing viable skin can be divided into short-term and long-term techniques. As a large variation in protocols have been published for storage of skin grafts and those have been extensively reviewed [54,74,74-76], only methods useful for in vitro testing are discussed in this section. As a consequence, some protocols that are recommended by guidelines and standards, are not

Type Method Treatment duration Tissue integrity Tissue viability Reproducibility Mechanical _Cutting (cryotome) 24 hrs 0 - -Cantharidin 4.5 hrs - -

-Ionic change Ammonia 45 min - - 0

Enzymatic digestion Trypsin 2-24 hrs + + +