European Cells&and

MW Laschke MD Materials
Menger Vol. 32 2016 (pages 202-215) DOI: 10.22203/eCM.v032a13 Dorsal skinfoldISSN 1473-2262
chamber model

THE DORSAL SKINFOLD CHAMBER: A VERSATILE TOOL FOR PRECLINICAL

RESEARCH IN TISSUE ENGINEERING AND REGENERATIVE MEDICINE

M.W. Laschke* and M.D. Menger

Institute for Clinical & Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany

Abstract Introduction

The dorsal skinfold chamber is a rodent model for non- Regenerative medicine is a rapidly growing research field,
invasive microcirculatory analyses of striated muscle and including complex in vivo processes such as the wound
skin tissue throughout an observation period of 2-3 weeks. healing cascade (Reinke and Sorg, 2012), acute and chronic
In combination with intravital fluorescence microscopy, tissue responses to medical implants (Anderson et al.,
this model allows the quantitative assessment of dynamic 2008) and the vascularisation and integration of skin grafts
processes such as inflammation, angiogenesis, vascular and surgical flaps (Hallock and Morris, 2011). All of these
remodelling and microcirculation. Accordingly, the dorsal processes are characterised by a highly dynamic interaction
skinfold chamber is increasingly used for preclinical of signalling molecules, cells and the microcirculation.
research in tissue engineering and regenerative medicine. Hence, they cannot be simulated in vitro or in silico.
This includes studies on biocompatibility, vascularisation Accordingly, there is a strong need for sophisticated animal
and incorporation of medical implants and artificial tissue models, which allow standardised analyses under in vivo
constructs. Moreover, the chamber implantation procedure conditions. This is a major prerequisite for the successful
has been modified to analyse primary and secondary wound transfer of novel regenerative approaches into clinical
healing as well as revascularisation and blood perfusion practice.
of dermal substitutes, skin grafts and myocutaneous The dorsal skinfold chamber is such a model. It
flaps. Hence, the dorsal skinfold chamber model does not has been broadly used to investigate microcirculation
only provide deep insights into fundamental regenerative physiology (Funk et al., 1983; Gerstberger et al., 1988),
mechanisms but also represents a versatile tool for the microcirculatory effects of anaesthesia (Franke et al.,
development of novel therapeutic strategies. 1982; Franke and Endrich, 1983), bacteria-endothelial cell
interaction (Pappelbaum et al., 2013), inflammation and
sepsis (Hoffmann et al., 2004; Hillgruber et al., 2014; de
Keywords: Tissue engineering, angiogenesis, Miranda et al., 2015), haemorrhagic shock (Ortiz et al.,
inflammation, microcirculation, biocompatibility, scaffold, 2014), thrombosis and thrombolysis (Boulaftali et al.,
skin, dermal substitute, wound healing, flap. 2012; Ampofo et al., 2015a), ischaemia and reperfusion
(Menger et al., 2003; Ampofo et al., 2016), tissue and organ
transplantation (Funk et al., 1986; Bordel et al., 2006;
Wittig et al., 2013), endometriosis (Laschke and Menger,
2007; Nenicu et al., 2014), hereditary haemorrhagic
telangiectasia (Garrido-Martin et al., 2014) as well as
tumour biology and therapy (Debergh et al., 2010; Baron et
al., 2011). The model is based on the chronic implantation
of an observation chamber (Fig. 1) on the dorsal skinfold
of rats, hamsters or mice. This provides continuous access
to striated muscle, i.e. panniculus carnosus, subcutis and
cutis for repetitive, non-invasive intravital microscopic
analyses throughout an observation period of 2-3 weeks
(Menger et al., 2002).
During the last two decades, an increasing number
of studies indicates that the dorsal skinfold chamber is a
versatile tool for preclinical research in tissue engineering
and regenerative medicine. This model is frequently used
for the evaluation of medical implants and engineered
*Address for correspondence: tissue constructs, because they can be easily implanted
Matthias W. Laschke, M.D., Ph.D. into the chamber and because their biocompatibility and
Institute for Clinical & Experimental Surgery vascularisation can be repetitively studied by means
Saarland University of intravital fluorescence microscopy. In addition, the
D-66421 Homburg/Saar, Germany chamber implantation and tissue preparation procedure
Telephone number: +49 6841 162 6554 have been modified in different ways to set ideal conditions
Fax number: +49 6841 162 6553 for the in vivo analysis of primary and secondary wound
Email: matthias.laschke@uks.eu healing as well as revascularisation and blood perfusion of

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MW Laschke & MD Menger Dorsal skinfold chamber model

Fig. 1. Mounted dorsal skinfold chamber for mice.

skin grafts, dermal substitutes and myocutaneous flaps. In or the in situ staining of leukocytes with rhodamine 6G to
this review, we provide an overview of these approaches study their interaction with the microvascular endothelium
and novel insights into fundamental regenerative processes, during inflammation (Ampofo et al., 2015b). Apoptotic
which have been achieved by these approaches. cells can be identified by an increased nuclear chromatin
condensation and fragmentation by topical bisbenzimide
staining (Vollmar et al., 2001).
Medical implants and engineered tissue constructs The dorsal skinfold chamber model is frequently
used for implant research, because the cover glass of
The preparation of the dorsal skinfold chamber has been the observation window can be temporarily removed for
described previously in detail (Laschke et al., 2011). It the implantation of biomaterials and engineered tissue
involves the implantation of two symmetrical titanium constructs onto the panniculus carnosus muscle (Laschke
or polymer frames onto the back of the anaesthetised et al., 2011). For this purpose, the animals are allowed
animal. During the implantation of the frames, one layer to recover from the surgical trauma of the chamber
of cutis, subcutis and panniculus carnosus muscle is preparation procedure for 48-72 h prior to implantation
carefully removed in a circular area at the front side of to guarantee physiological conditions for the analysis
the chamber, which serves as the observation window for of the inflammatory and angiogenic host tissue response
subsequent analyses. Hence, this window finally includes to the implants. The size of the implants is restricted to
the contralateral layer of panniculus carnosus muscle, ~3 × 3 × 1 mm for an air-free closure of the chamber
subcutis and skin (Fig. 2a). The exposed tissue is attached and to enable analyses of the implants themselves and
to a cover glass, which is fixed with a snap ring in the the surrounding tissue. Accordingly, the implants cannot
chamber frame. be tested in a size, which is normally needed for clinical
The dorsal skinfold chamber bears the major advantage applications. Moreover, the host tissue inside the chamber
that it can be horizontally positioned under a microscope is not always identical to the host tissue at the orthotopic
for repetitive, non-invasive intravital microscopy in implantation site of certain biomaterials. Hence, this
transillumination or fluorescence epi-illumination different environment may not allow the direct comparison
technique. After the application of different fluorescent of the obtained results with findings from orthotopic
dyes, the epi-illumination approach allows high-resolution studies. On the other hand, the experimental setting of the
imaging and computer-assisted quantitative analyses of dorsal skinfold chamber allows the in vivo evaluation of
distinct microvascular, cellular and molecular mechanisms. many different types of implants under highly standardised
Typical examples are the visualisation of individual conditions.
microvessels by intravenous injection of the plasma marker The use of biomaterials in regenerative medicine
fluorescein isothiocyanate (FITC)-labelled dextran for always raises the question of biocompatibility and
the assessment of microhaemodynamic parameters and vascularisation. A good biocompatibility is an essential
angiogenic processes (Ring et al., 2011a; Feng et al., 2014) prerequisite for a safe long-term implantation and function

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Fig. 2. Different applications of the dorsal skinfold chamber model for analyses in the field of tissue engineering
and regenerative medicine, as described in the corresponding sections of this review article. The schematic cross-
sections display a dorsal skinfold chamber for the analysis of medical implants and engineered tissue constructs (a),
healing of primary (b) and secondary wounds (c), dermal substitutes (d), skin grafts (e) and myocutaneous flaps (f).

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MW Laschke & MD Menger Dorsal skinfold chamber model

of biomaterials without inducing severe local or systemic of host blood vessels into large three-dimensional tissue
inflammatory side effects (Shard and Tomlins, 2006). constructs is not sufficient to guarantee the survival of
A rapid and sufficient vascularisation is important for seeded cells. Accordingly, there is a clear trend towards
the adequate incorporation of medical implants into the prevascularisation strategies (Laschke and Menger, 2016).
surrounding host tissue and the survival of cells within These strategies focus on the generation of intrinsic
tissue constructs (Rouwkema and Khademhosseini, 2016). microvascular networks within tissue constructs, which
By means of the dorsal skinfold chamber model it has been are rapidly blood perfused by forming interconnections
shown that the two factors are crucially dependent on each with the surrounding host microvasculature after
other. Biomaterials which do not induce any inflammatory implantation. This can be achieved by seeding scaffolds
host tissue response also exhibit a poor vascularisation with adipose-derived MSC spheroids, which promote more
after implantation (Laschke et al., 2010). In fact, vascular effectively the simultaneous development of microvascular
ingrowth into implanted biomaterials is only promoted in networks at different locations inside the implants when
the presence of a temporary moderate tissue inflammation compared to seeded single MSCs (Laschke et al., 2013).
(Rücker et al., 2006; Rücker et al., 2008; Laschke et Another promising approach is the seeding of scaffolds
al., 2014). In contrast, a prolonged severe inflammation with microvascular fragments, which are enzymatically
inhibits angiogenesis at the implantation site (Rücker et al., isolated in large amounts from fat tissue and consist of
2006). This indicates that controllable pro-inflammatory fully functional arteriolar, capillary and venular vessel
conditions are beneficial for the incorporation process. segments (Laschke and Menger, 2015). After implantation,
The biological reaction to biomaterials is crucially the microvascular fragments reassemble to blood-perfused
determined by the physico-chemical characteristics of microvascular networks and, thus, markedly contribute
their surface, their porosity and degradation kinetics. to the rapid vascularisation of implanted tissue constructs
Surface activation by gas plasma treatment improves (Laschke et al., 2012).
vascular ingrowth into polymer-based and allogenic
bone implants in mouse dorsal skinfold chambers (Ring
et al., 2007; Ring et al., 2011b). However, dependent on Primary wound healing
the used raw material this type of treatment may also
generate nanorough surfaces which, for instance, impair For the analysis of primary wound healing, the dorsal
cell attachment and vascularisation of porous polyethylene skinfold chamber is prepared as described above,
(Medpor®) (Laschke et al., 2016). Druecke et al. (2004) containing one layer of panniculus carnosus muscle,
found that poly(ether ester) block-copolymer implants subcutis and skin (Fig. 2a). Subsequently, these layers
with large pores of 250-300 µm exhibit more blood vessels are carefully cut with a scalpel over a length of 7 mm
after implantation when compared to implants with smaller in the centre of the observation window and the edges
pore sizes. In line with these findings, Abshagen et al. of the resulting incisional wound are sutured with 9/0
(2009) further detected a stronger angiogenic response to Nylon at 1 mm intervals (Figs. 2b and 3a). The tissue
the synthetic bone grafting substitute NanoBone® when preparation is kept moist with physiological saline and
implanted as widely spaced small granules in comparison sealed with a cover glass, which provides continuous
to large solid plates. Biodegradation of biomaterials has access to the healing wound throughout an observation
been shown to induce angiogenesis at the implantation site period of 2-3 weeks. This time period is sufficient to study
(Ghanaati et al., 2009). Of interest, degraded areas within the entire incisional wound healing process, including the
implants may even enable a guided neovascularisation early inflammatory phase as well as the formation of a
(Laschke et al., 2007). vascularised granulation tissue surrounding the sutures.
Beside the characterisation of different biological Importantly, intravital fluorescence microscopy enables
responses to distinct biomaterial properties, the dorsal the visualisation of individual vascular sprouts and newly
skinfold chamber model has been used to assess the formed microvessels within the wound margins. Thus,
efficiency of novel vascularisation strategies for tissue this approach does not only provide detailed insights into
engineering. A common approach is the coating of microvascular network morphology and remodelling but
scaffold biomaterials with different pro-angiogenic also into microhaemodynamic changes during incisional
factors, including vascular endothelial growth factor wound healing.
(VEGF) (Lindhorst et al., 2010; Strieth et al., 2010), Contaldo et al. (2012) prepared incisional wounds in
sphingosine 1-phosphate (Sefcik et al., 2008), human dorsal skinfold chambers of mice genetically depleted
host defence peptide LL37 (Steinstraesser et al., 2006), of apolipoprotein E (ApoE-/-), which were repetitively
phthalimide neovascular factor (PNF)-1 (Wieghaus et al., treated with radial pressure waves. They found that this
2008) and stromal derived factor (SDF)-1α (Krieger et type of treatment significantly improved primary wound
al., 2016). Moreover, scaffolds are vitalised with growth healing in the hypercholesterolaemic mice by promoting
factor-secreting cells, such as mesenchymal stem cells functional angiogenesis and enhancing tissue organisation.
(MSCs) (Kampmann et al., 2013; Schumann et al., 2014), They also observed an increased expression of caspase-3,
osteoblasts (Schumann et al., 2009; Tavassol et al., 2010) proliferating cell nuclear antigen and endothelial nitric
or chondrocytes (Ehrmantraut et al., 2012), to induce oxide synthase (eNOS) in the early phase of the healing
angiogenesis at the implantation site. However, because the process, indicating that the treatment with radial pressure
physiological growth rate of microvessels is not faster than waves may facilitate the linear progression of the wound
~5 µm/h (Utzinger et al., 2015), the angiogenic ingrowth healing cascade. In the identical experimental setting,

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Elsherbiny et al. (2012) demonstrated that repetitive

application of erythropoietin (EPO) reverses microvascular
dysfunction in hypercholesterolaemic mice (Figs. 3b-g).
EPO treatment stimulated angiogenesis and increased
the capacity of wound endothelial cells to produce nitric
oxide (NO) through enhanced EPO receptor and eNOS
expression. This resulted in an improved blood perfusion
of the wound margins comparable to that in normal healing
wild-type animals. Accordingly, systemic EPO therapy may
represent a promising strategy to restore the angiogenic
tissue response of patients with microangiopathy during
wound healing.

Secondary wound healing

In the secondary wound healing model, the two chamber

frames are first completely fixed to the dorsal skinfold
of the animals without tissue preparation. Subsequently,
a round shaped full-thickness dermal wound with a
defined diameter of 3-4  mm is created in the centre of
the observation window by removing one layer of cutis,
subcutis and panniculus carnosus muscle using a biopsy
punch and microsurgical instruments (Fig. 2c). The wound
is rinsed with physiological saline and the observation
window is closed with a cover glass to study wound healing
under wet conditions. However, it is also possible to omit
the cover glass for the analysis of dry wounds as in other
well established wound healing models such as the ear
of the hairless mouse (Kuehnl et al., 2013; Yousefi et al.,
2014). In both cases, it is mandatory to depilate thoroughly
the skin of the animals during the preparation procedure
to guarantee ideal imaging conditions without interfering
growing fur in the observation window. Alternatively,
hairless mice may be used. Ideally, they should exhibit an
intact immune system, such as SKH1 mice (Benavides et
al., 2009), to exclude severe effects of immunosuppression,
as known for nude mice, on inflammation and angiogenesis
during wound healing.
The closure of wounds in loose-skinned rodents
is typically associated with wound contraction by the
panniculus carnosus muscle and myofibroblasts (Horan Fig. 3. Dorsal skinfold chamber model for the study of
et al., 2005; Davidson et al., 2013). The prevention of primary wound healing according to Elsherbiny et al.
this contraction is one of the major prerequisites for the (2012). (a) Photomacroscopic image of an incisional
establishment of reliable models adequately reflecting wound in the centre of the observation window.
secondary wound healing by granulation tissue formation (b-g) Intravital fluorescence microscopy (contrast
in humans. In the dorsal skinfold chamber model this is enhancement by 5 % FITC-labelled dextran in epi-
achieved by spanning the double layer of skin between the illumination) of incisional wounds in dorsal skinfold
chamber frames. After positioning the observation window chambers of vehicle-treated (b,d,f) and EPO-treated
under a stereomicroscope, continuous wound closure can ApoE-/- mice (1000  U/kg body weight i.p.; c,e,g) at
be quantitatively analysed by planimetric measurements day 3 (b,c), 7 (d,e) and 9 (f,g) post-wounding. Note the
of epithelialised wound areas (Figs. 4a-f). Intravital higher microvessel density around the 9/0 Nylon suture
fluorescence microscopy further provides detailed insights (black) in the EPO-treated animal when compared to the
into microvascular changes during the wound healing control. Reproduced from Elsherbiny et al. (2012) with
process. Sorg et al. (2007) found that the skin in the permission from Elsevier.
observation window of the chamber exhibits an outer radial
pattern of vessels which supply an inner circular ring of
newly formed vessels at the wound margin. In the course
of inwards directed epithelialisation, the inner circular
ring progressively grows as a moving vascularisation front

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to the wound centre and finally regresses after complete this model can only be used to study the early inflammatory
wound closure (Sorg et al., 2007). and angiogenic host tissue response to dermal substitutes.
In the search for novel therapeutic options to improve This, however, is not necessarily a disadvantage, because
wound healing, systemic application of repetitive low under clinical conditions the early vascularisation and
doses but especially single high doses of EPO have been incorporation of the implants may particularly determine
shown to improve secondary wound healing in the dorsal the quality of healing and the risk of wound infection.
skinfold chamber of hairless mice (Sorg et al., 2009). Thus, the initial post-implantation phase is also of major
This treatment accelerates wound epithelialisation and interest in preclinical studies. An advantage of the dorsal
stimulates maturation of newly developing microvascular skinfold chamber model is the fact that the implanted
networks. In contrast, repetitive high doses of EPO dermal substitutes are protected by the cover glass from
induce massive erythrocytosis, resulting in rheological exsiccation and manipulation by the animals (Michael et
malfunction, impaired vessel maturation and delayed al., 2013a). There is also no need for additional wound
wound closure. Another interesting approach to promote dressings and their regular changes, which may markedly
dermal regeneration is the systemic pretreatment with affect the incorporation process by damaging the tissue
unmethylated CpG oligodeoxynucleotides (ODNs) preparation and stressing the animals.
(Hergert et al., 2013). ODNs stimulate the immune system Michael et al. (2013a) implanted a dermal construct
by binding to Toll-like receptor-9. In healing wounds, comprising a collagen type I gel on top of a Matriderm®
this supports the shift from M1-polarised inflammatory layer into 6 mm full-thickness skin wounds within the
macrophages towards regenerative M2-polarised observation window of mouse dorsal skinfold chambers.
macrophages, attracts fibroblasts and keratinocytes and, Histological and immunohistochemical analyses after 11 d
thus, improves tissue-remodelling processes and wound revealed that the implants were invaded by fibroblasts and
epithelialisation. started to epithelialise from the wound edges. However,
It is further possible to investigate muscle wound they did not yet exhibit any ingrowing microvessels. In a
healing in dorsal skinfold chambers by creating lesions follow-up study (Michael et al., 2013b) the Matriderm®
of the panniculus carnosus muscle in the centre of the layer was coated with 20 layers of fibroblast-containing
observation window. This approach was used by Machado collagen and 20 layers of keratinocyte-containing collagen
and Mitchell (2011) to study endothelial barrier function in by means of laser-assisted bioprinting. The two cell
pre-existing vessels as well as angiogenic plexus and blind- types survived the printing and implantation procedure
ended vessels in discrete wounds. They found a relationship and markedly contributed to the formation of a multi-
between temporal changes in macromolecular flux and the layered epidermis with an underlying corneal layer and
morphology of maturating vascular segments. In addition, collagen deposition into the matrix. This was associated
they performed longitudinal confocal microscopic analyses with an improved vascularisation of the implants. These
of perfused vascular segments to create a mathematical findings indicate that laser-assisted bioprinting represents
model of angiogenesis during wound healing (Machado a promising approach for the tissue engineering of skin
et al., 2011). Finally, Langer et al. (2016) reported that constructs with improved biological properties when
vascularisation and closure of panniculus carnosus lesions compared to cell-free dermal substitutes.
are affected in diabetic mice when compared to wild-type Kijanska et al. (2016) recently used the dorsal skinfold
controls. This study nicely demonstrates that the skinfold chamber model to analyse in vivo the integration and
chamber model is not only suitable for the testing of novel vascularisation of SERI® Surgical Scaffold, which is a
wound therapies in healthy animals but also to investigate bioresorbable, silk-derived multifilament scaffold for
the mechanisms of disturbed wound healing under different soft tissue support (Gross et al., 2014). By means of
pathophysiological conditions by means of appropriate immunohistochemical analyses and corrosion casting
mouse strains. they found that the scaffold induces an inflammatory host
tissue response, which promotes the rapid ingrowth of a
vascularised granulation tissue in the scaffold pores. These
Dermal substitutes results are in line with clinical studies reporting an excellent
in vivo performance of SERI® Surgical Scaffold in breast
The chamber preparation for the analysis of secondary reconstruction, circumferential abdominoplasty and lower
wound healing is also suitable for the evaluation of dermal body lift (Kornstein, 2014; Fine et al., 2015).
substitutes and tissue engineered skin. For this purpose,
the full-thickness skin wound is usually created with a
larger diameter of 6-7 mm for the press fit implantation Skin grafts
of the substitutes (Fig. 2d). Nonetheless, this wound
area is still rather small when compared to the common For the analysis of skin graft revascularisation, a dorsal
model of dorso-lateral full-thickness wounds without a skinfold chamber is prepared in a first step. After a recovery
chamber allowing the assessment of implants with a size period of 3 d, the cutis and parts of the subcutis are removed
of up to 2 × 3 cm (Rasmussen et al., 2010). Moreover, the in a circular area of 7 mm in diameter from the back of the
implants can only be analysed throughout an observation chamber, resulting in a wound bed consisting of panniculus
period of 2-3 weeks, because the elasticity of the dorsal carnosus muscle and subcutaneous tissue (Lindenblatt et
skinfold decreases over time which can lead to tilting of the al., 2008; Lindenblatt et al., 2010) (Figs. 2e and 5a-d). A
chamber and perfusion failure of the tissue. Accordingly, full-thickness skin graft of identical size is then excised

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MW Laschke & MD Menger Dorsal skinfold chamber model

Fig. 4. Dorsal skinfold chamber

model for the study of secondary
wound healing according to Sorg
et al. (2007). Photomacroscopic
images of the skinfold chamber
prior to day 0pre (a) and directly
after wounding 0post (b), as well as
at day 3 (c), 6 (d), 9 (e) and 12 (f),
displaying the continuous process
of wound closure with complete
epithelialisation at day 12 (dotted
lines: wound border; broken lines:
border of non-epithelialised wound
area). Reproduced from Sorg et al.
(2007) with permission from John
Wiley and Sons.

from the groin of the animal and fixed in the defect with reperfusion phase (Lindenblatt et al., 2010). By suturing
polypropylene 8-0 sutures. The observation window of the skin grafts from transgenic green fluorescent protein
back side is finally closed with a cover glass. (GFP)-positive donor mice into dorsal skinfold chambers
The major advantage of this modified dorsal skinfold of GFP-negative wild-type mice and vice versa, Calcagni
chamber model is the fact that it enables simultaneous et al. (2011) further found that 68 % of existing graft
intravital fluorescent microscopic analyses of the wound capillaries are replaced over time by ingrowing vessels
bed from the front of the chamber and the skin graft from the from the wound bed using the old vessels as channels.
back of the chamber during the first 10 d after engraftment Accordingly, they suggested that comparably to skin grafts,
(Lindenblatt et al., 2008). Thus, it allows not only the fabricated skin substitutes should also provide preformed
visualisation of typical morphological characteristics of the vascular channels to improve their vascularisation after
angiogenic process, such as the formation of capillary buds implantation.
and sprouts, but also the assessment of blood perfusion in In plastic surgery, surplus harvested skin grafts are
individual microvessels of the grafts and the host tissue. usually stored at 4-6 °C in saline for several days before
Lindenblatt et al. (2008) could demonstrate that skin grafts transplantation. Knapik et al. (2014) analysed how this
induce angiogenesis in the wound bed with ingrowth type of preservation influences the engraftment of freshly
of newly developing microvessels into the grafts and harvested human full-thickness and split-thickness skin
subsequent connection to the existing vasculature after grafts in severe combined immunodeficiency (SCID)
3 d. Knapik et al. (2012) showed that this connection is mice. They found that host capillary ingrowth and graft
mediated by matrix metalloproteinase MT1-MMP, which vessel degeneration were not affected by cold storage.
is specifically expressed at the tips of ingrowing sprouts, However, the warming up of the grafts in the wound bed
stimulating their proliferation and lysing the existing caused significant tissue injury, which was not observed
graft capillaries in order to connect to them. The onset of prior to transplantation. These findings indicate that skin
blood perfusion, in turn, induces a temporary angiogenic tissue is protected from damage under cold conditions,
response within the grafts’ capillaries between days 3 and most likely due to a reduced cell metabolism. Nonetheless,
8. This most likely represents a reaction to accumulating tissue damage is higher in cold stored skin grafts, which is
angiogenic growth factors in the hypoxic tissue during the disclosed in the rewarming phase after transplantation.

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Fig. 5. Dorsal skinfold chamber model for the study of skin graft revascularisation according to Lindenblatt et al.
(2010). (a) From the front of the chamber, the muscle wound bed (panniculus carnosus) and the larger subcutaneous
vessels are visible. (b) The microcirculation of the skin graft can be visualised from the back of the chamber. (c,d)
Intravital fluorescence microscopy (contrast enhancement by 5 % FITC-labelled dextran in epi-illumination) shows
a strong angiogenic response of the wound bed after 48 h (c), resulting in reperfusion of the graft capillaries after
72 h (d). Reproduced from Lindenblatt et al. (2010) with permission from Wolters Kluwer Health, Inc.

Myocutaneous flaps frame is temporarily placed on the double-layered skinfold

to outline the flap on the skin with its distal 2 mm extending
Ichioka et al. (2002) demonstrated for the first time the to the contralateral side (Fig. 6a). The deep circumflex iliac
suitability of skinfold chambers for the analysis of flap artery and the lateral thoracic artery are then transected
microcirculation. They implanted a newly designed small during the elevation of the flap (Fig. 6b). Subsequently,
chamber in a large bipedicled island flap on the dorsum the top edge of the flap, consisting of cutis, subcutis and
of mice. To induce partial flap necrosis, the vascular panniculus carnosus muscle, is fixed to the chamber’s
pedicles of the island flap were temporarily clamped to back side frame (Fig. 6c). The lateral sides are sutured
induce global ischaemia and reperfusion. In contrast, back to the surrounding skinfold (Fig. 6d). In addition,
Harder et al. (2004) combined the mouse dorsal skinfold the overlaying skin layer of the opposite side of the flap
chamber with a laterally based random pattern flap with is removed, which allows the direct visualisation onto the
a width-to-length ratio of 15 × 11 mm. Importantly, this elevated flap tissue (Fig. 6d). Finally, the preparation is
flap undergoes persistent ischaemia and spontaneously covered with the front side frame of the chamber (Fig.
develops 50 % necrosis if kept untreated. Accordingly, it 6e) and the observation window is closed with a cover
has been frequently used to study pathological mechanisms glass that provides continuous access to the flap tissue for
in critically ischaemic myocutaneous tissue and to evaluate repetitive intravital microscopy (Figs. 2f and 6f).
novel therapeutic strategies to improve flap perfusion and Analyses of the microcirculation and partial oxygen
survival (Harder et al., 2014). For this purpose, repetitive tension in different zones of the myocutaneous flap
microcirculatory in vivo measurements in different regions revealed that the critical ischaemia-induced necrosis
of the flap can be combined with immunohistochemical and does not demarcate sharply from the adequately perfused
molecular biological analyses of the flap tissue. tissue at the flap basis (Harder et al., 2005a). Between the
For the preparation of the random pattern flap, the back two zones a fringe of tissue with vascular remodelling
of the animal is depilated and the chamber’s back side develops a distally adjacent falx lunatica lacking nutritive

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Fig. 6. Dorsal skinfold chamber model for the study of myocutaneous flaps according to Harder et al. (2004). (a) Flap
outlined on the skin (width/length 15 mm/11 mm) with its distal 2 mm extending to the contralateral side. To study
the back side of the flap tissue through the observation window, additional tissue (hatched area) has to be removed.
(b) Elevation of the laterally based flap with a distinct, however randomly arranged vascular architecture. (c,d) Flap
extended on the back side of the frame (c) sutured back into the dorsal skin (d) to guarantee tightness. (e) Fully mounted
chamber with cover glass and brown sealing band exceeding the device both laterally and on top immediately after
the operation. (f) Mouse after implantation of dorsal skinfold chamber according to controls without flap elevation.
(g) Observation window instantly after the preparation, displaying an intact microvasculature (control animal). (h,j)
Observation window at day 1 (h) and day 3 (j) after flap elevation, demonstrating a clear line of demarcation which
moves toward the flap’s base. Reproduced from Harder et al. (2004) with permission from Elsevier.

blood perfusion and solely surviving by oxygen diffusion Conclusions

(Harder et al., 2005a). Additional studies have shown that
the perfusion and survival of the flap can be improved The herein described studies indicate that the dorsal
by different therapeutic approaches. These include local skinfold chamber is a versatile tool for regenerative
heat preconditioning (Harder et al., 2005b), selective medicine research. The major advantage of this model is
blockade of endothelin-B receptor (Wettstein et al., the direct access to the analysed tissues and implants, which
2007) as well as pharmacological treatment with EPO are protected from manipulation by the animals within
(Rezaeian et al., 2008; Harder et al., 2009; Rezaeian the observation window. In combination with intravital
et al., 2013), n-acetylcysteine (Bächle et al., 2011) and fluorescence microscopic techniques this allows repetitive,
ghrelin (Rezaeian et al., 2012). Promising results of these non-invasive analyses of dynamic regenerative processes
preclinical studies have encouraged the conduction of first with quantitative measurements of functional parameters
translational studies in the clinical setting. For instance, such as microvascular perfusion or leukocyte-endothelial
Mehta et al. (2013) demonstrated in a prospective non- cell interactions with a high spatial and temporal resolution.
randomised trial including 50 patients that local heat However, the analyses are restricted to an observation
preconditioning reduces skin necrosis and hospitalisation period of 2-3 weeks after chamber preparation, because the
length following skin sparing mastectomy and immediate elasticity of the dorsal skinfold is lost over time. Moreover,
breast reconstruction. the imaging quality within the observation window may

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progressively decrease due to repetitive applications of Ampofo E, Widmaier D, Montenarh M, Menger

fluorescent dyes and their increased vascular leakage under MD, Laschke MW (2016) Protein kinase CK2 regulates
angiogenic or inflammatory conditions. Accordingly, the leukocyte-endothelial cell jnteractions during ischemia
dorsal skinfold chamber is particularly a model for the and reperfusion in striated skin muscle. Eur Surg Res 57:
detailed analysis of early biological events, which crucially 111-124.
determine the long-term outcome of regenerative processes Anderson JM, Rodriguez A, Chang DT (2008) Foreign
such as biomaterial integration (Sclafani et al., 1997; Naik body reaction to biomaterials. Semin Immunol 20: 86-100.
et al., 2007). Due to the fixation of the skin tissue between Baron VT, Welsh J, Abedinpour P, Borgström P (2011)
the two chamber frames, it is also well suited for the study Intravital microscopy in the mouse dorsal chamber model
of wound healing, engraftment of dermal substitutes for the study of solid tumors. Am J Cancer Res 1: 674-686.
and flap survival without contraction of the underlying Bächle AC, Mörsdorf P, Rezaeian F, Ong MF, Harder Y,
panniculus carnosus muscle, which represents a typical Menger MD (2011) N-acetylcysteine attenuates leukocytic
pitfall of other models in loose-skinned rodents. inflammation and microvascular perfusion failure in
It has to be further considered that the given dimensions critically ischemic random pattern flaps. Microvasc Res
of the dorsal skinfold chamber markedly limit the size 82: 28-34.
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much larger in the clinical setting. On the other hand, this VE, Kusewitt DF (2009) The hairless mouse in skin
allows their examination under standardised conditions research. J Dermatol Sci 53: 10-18.
without biases due to individual size adjustments. Bordel R, Laschke MW, Menger MD, Vollmar B
Moreover, they can be manipulated in the chamber without (2006) Nicotine does not affect vascularisation but inhibits
affecting the general condition of the animal. This may growth of freely transplanted ovarian follicles by inducing
be particularly advantageous for studies focusing on granulosa cell apoptosis. Hum Reprod 21: 610-617.
hypo- and hyperthermic preconditioning or the evaluation Boulaftali Y, Lamrani L, Rouzaud MC, Loyau S,
of pharmacological compounds, which can be locally Jandrot-Perrus M, Bouton MC, Ho-Tin-Noé B (2012) The
administered into the observation window. By pressing a mouse dorsal skinfold chamber as a model for the study of
silicone pad on the cutis of the back side of the window thrombolysis by intravital microscopy. Thromb Haemost
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Taken together, these characteristics of the dorsal 237-245.
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perfused musculocutaneous flaps. Langenbecks Arch Surg the testing is performed in a highly vascularised area.
392: 331-338. How do results translate when it comes to orthotopic
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Sefcik LS, Price RJ, Paige MA, Brown ML, Botchwey ischaemic?
EA (2008) Expansion of microvascular networks in vivo Authors: The reviewer is correct that the size of biomaterial
by phthalimide neovascular factor 1 (PNF1). Biomaterials implants in the dorsal skinfold chamber is restricted to
29: 4698-4708. ~3  ×  3  ×  1  mm. In addition, the host tissue inside the
Wittig C, Laschke MW, Scheuer C, Menger MD chamber is well vascularised and, thus, provides optimal
(2013) Incorporation of bone marrow cells in pancreatic conditions for cell survival and vascular ingrowth, which
pseudoislets improves posttransplant vascularisation and are not always guaranteed at the orthotopic implantation
endocrine function. PLoS One 8: e69975. site of clinically applied biomaterials. Nonetheless, the
Yousefi S, Qin J, Dziennis S, Wang RK (2014) dorsal skinfold chamber model bears the major advantage
Assessment of microcirculation dynamics during that it provides highly standardised conditions for the in
cutaneous wound healing phases in vivo using optical vivo characterisation of different biomaterials without
microangiography. J Biomed Opt 19: 76015. biases due to individual size adjustments. Hence, results
obtained in different studies can be directly compared with
each other. Beyond, this model even allows the testing of
Discussion with Reviewer biomaterials under clearly defined ischaemic conditions
by pressing a silicone pad on the cutis of the back side of
Marietta Herrmann: The skinfold chamber has been the chamber window.
widely applied to study neovascularisation of biomaterials
and TE constructs. The dimensions of the constructs are Editor’s note: The Scientific Editor responsible for this
however quite different from the target implant. Moreover, paper was Juerg Gasser.

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