2.1.

Study Participants and Study Protocol

Six $(n=6)$ individuals aged between 22 and $42\text{years}$ with no history of skin diseases participated in the study after their written consent was obtained. All participants were recruited from the Department of Dermatology, Charité University Medicine, Berlin. All research was conducted according to the Declaration of Helsinki principle.

2.2.

Methods of Epidermal Wounding

At day 0, two suction blisters were induced on the untreated volar forearms of the study participants. The blisters were established using the tubes of syringes pressed upside down onto the skin. Next, the needles were connected to a vacuum pump applying a negative pressure at $0.3\text{bar}$ for $60\min$ (Fig. 1 ). Following the generation of the suction blisters, the vacuum pump was disconnected and the roof of the blister, corresponding to the full epidermal thickness was removed.

Fig. 1

Induction of suction blisters for the study of epidermal wounds. (a) Tubes of syringes pressed upside-down onto the skin. Next, the needle was connected to a vacuum pump applying a negative pressure at $0.3\text{bar}$ for $60\min .$ (b). After the generation of the suction blisters, the vacuum pump was disconnected, and the roof of the blister representing the epidermis was removed.

2.3.

Treatment of Epidermal Wounds

After the removal of the epidermis, one of the two established wound sites on the forearm of the participants was treated twice a day with the wound ointment over a time period of $8\text{days}$ . The remaining wound site remained untreated, subsequently serving as control site. During the entire duration of the study, all wound sites were covered with a self-adhesive wound dressing made of a soft nonwoven fabric as support material, with an absorbent wound dressing pad made of 100% absorbent cotton wool, covered with a nonadherent microgrid wound-contact layer. The wound dressing was changed twice a day after the investigation of the wound site. The ointment used for the studies consisted of dexpanthenol 5%, cera alba, paraffinum liquidum, aqua, cetyl alcohol, stearyl alcohol, almond oil, lanolin alcohol, petrolatum, glyceryl oleate, and ozokerite.

2.4.

Evaluation of Epidermal Wound Healing

The time course of tissue repair was documented on days 1, 3, 5, 7, and 8 by FLSM followed by descriptional image analysis, combined with a photographical documentation of the clinical presentation over a time period of 8 consecutive days.

In order to analyze the progression of tissue repair, the process of wound healing was divided into three phases. Phase 1 was defined as the time point after the removal of the epidermal blister roof characterized by inflammation and wound exudation. In phase 2, the formation of the first layer of corneocytes was observed. Phase 3 describes the end of tissue repair with all reemerged layers of corneocytes and a reestablished skin barrier. The classification of the healing wounds into these three phases allowed the documentation of differences in the advancement of tissue repair between the study groups—i.e., those treated with topical ointment versus the group of untreated wounds.

We determined the specific phase of wound healing for all six study participants on days 1, 3, 5, 7, and 8, and the means of the wound healing phase were calculated for each group for all investigated time points. To compare means of untreated and treated wounds, Student $t$ -tests for paired data were performed.

2.5.

Fluorescence Laser Scanning Microscopy (FLSM)

A commercially available fluorescence laser scanning microscope (Stratum, OptiScan Ltd., Melbourne, Australia) was used in this study. The technical details of this technique have been summarized in former publications.⁸

Coherent laser light from a $50\text{-}\mathrm{mW}$ , air-cooled, $488\text{-}\mathrm{nm}$ , single-line argon ion laser is focused onto the proximal end of a single-mode optical fiber. By focusing the laser light onto the fiber tip, the laser light will be coupled into, and propagated along, the optical fiber. The laser light emerges from the distal end of the optical fiber and is focused by the objective lens onto the tissue. Light emanating from the same focal point within the specimen and collected by the objective lens will be focused back onto the distal end of the optical fiber, and propagated back toward the laser light source. Once the returning light emerges from the proximal end of the optical fiber, it is separated from the illumination path using a beamsplitter (or dichroic mirror), and focused onto a photomultiplier tube for detection. Light that emanates from a point in the specimen that is outside the focal point of the objective lens will not be focused onto the distal tip of the optical fiber, and therefore will not be propagated to the detector, thus optically isolating the focal plane from the surrounding structures.

Fluorescent molecules within the specimen are excited to an electronic singlet state by absorption of the energy from the illuminating photons. When the excited fluorophore returns to its ground state, energy is released in the form of a photon. The emitted photon is of lower energy than the photons used to excite the fluorophore molecule (therefore at a longer wavelength). Thus, the light collected from the focal point in the specimen by the objective lens comprises both laser light backscattered from tissue structures (same wavelength as illumination source) and the longer wavelength emitted from the excited fluorophore molecules. As the returning fluorescence is a different wavelength (color) than the illumination source, a dichroic mirror is used to separate the light returning from the sample path from the illumination path. A fluorescence barrier filter is also placed in the light path between the dichroic mirror and the photodetector to block any reflected laser light, thus permitting only the longer wavelength fluorescent signal to pass through to the detector for measurement.

To facilitate further miniaturization and simplification of the optical light path, the Stratum employs a fused, biconical tapered fiber-optic coupler in place of the dichroic mirror and single optical fiber. A fiber-optic coupler is made from two lengths of optical fiber that are twisted together in the middle and heated so that they fuse together, resulting in four “arms.” When light travels down one arm toward the waist of the coupler, a proportion of the light can cross over into the other fiber at the coupler in a wavelength-dependent manner. Light will then emerge out of both exit arms of the coupler. The percentage of light emerging from each exit arm depends on the wavelength. This property is exploited to enable the signal returning from the specimen to be separated from the illumination path, thus eliminating the need for a dichroic beamsplitter, and reducing the total number of optical elements in the optical path of the instrument.

By scanning the focused point in a raster pattern over the specimen (point scanning), an array of point intensity measurements is collected from the area under investigation, and thus an image can be generated. In the Stratum, the focal point is moved over the field of view by physically moving the tip of the optical fiber within the scanner using $x$ and $y$ electromagnetic actuators. The imaging depth ( $z$ axis) in the sample is controlled by mechanically moving lens elements within the scanner to change the focal depth.

The maximum imaging depth in living tissue is up to approximately $300\mu \mathrm{m}$ and thereby restricted to the epidermis and superficial papillary dermis.

Fluorescence confocal microscopy relies on the differential distribution of endogenous (autofluorescence) or exogenous fluorescent molecules (fluorophores) within a tissue to produce contrast. Fluorescein sodium is a low molecular weight, water-soluble fluorescent contrast agent. Fluorescein has the advantages of high absorptivity, high quantum yield, and a peak maximum absorption, which closely matches the $488\mathrm{nm}$ spectral line of an argon ion laser. Topical and intradermal administration of fluorescein sodium (0.1% w/v solution) to the skin allows imaging of the superficial layers of the epidermis.

3.1.

Fluorescence Laser Scanning Microscopy Evaluation of Normal Skin

Overall, our findings are in agreement with former descriptions of normal healthy skin.⁶ While starting the imaging process from the skin surface and gradually progressing deeper along the $z$ axis, the first images are obtained from the stratum corneum [Fig. 2a ]. The anucleated cells are of polygonal shape and approximately $25\text{to}40\mu \mathrm{m}$ in size. The subjacent layer corresponds to the stratum granulosum and consists of two to four cell layers. Each cell is about $20\text{to}25\mu \mathrm{m}$ in size and presents with a central round to oval nucleus that appears as a well-demarcated structure of bright appearance within the cells. The cells of the stratum spinosum appear in a tight honeycombed pattern, with each cell measuring $15\text{to}20\mu \mathrm{m}$ in size separated from each other by distinct cell borders; nuclei appear as well-demarcated structures of bright appearance within the cells [Fig. 2b]. At the level of the basal and suprabasal layers, cells are seen as dark clusters, each measuring around $10\text{to}12\mu \mathrm{m}$ in size [Fig. 2c]. The dermoepidermal junction appears as round to oval rings of dark basal cells surrounding dermal papillae, which appear bright due to the homogenous distribution of the contrast agent in this layer [Fig. 2c].

Fig. 2

FLSM findings of normal skin. (a) FLSM morphology of the upper skin keratinocyte layer, the stratum corneum. Corneocytes appear as polygonal cells. Due to the anucleated nature of corneocytes, fluorescein uptake is visible only in the surrounding intercellular spaces. (b) Representative image of the next deeper layer, stratum spinosum, $25\text{to}50\mu \mathrm{m}$ below the stratum corneum. Spinous keratinocytes appear as dark oval-shaped cells, while the intercellular spaces appear bright. (c) Basal keratinocytes are surrounding each papillary structure and appear darker than surrounding keratinocytes. The center of each papilla appears bright due to the abundance of collagen. Scale bars: $50\mu \mathrm{m}$ .

3.2.

Fluorescence Laser Scanning Microscopy Evaluation of Epidermal Wounds

Figure 3a shows a representative clinical photograph of an epidermal wound in phase 1. The exudate is visible as slightly yellowish superficial deposit. This encrusted exudate can be visualized by FLSM [Fig. 3b] on the surface of the skin and the wound edge. By focusing the evaluation on the central wound bed, the basal layer may be observed, since the epidermis has been mechanically removed. Figure 3c shows a typical image of the suprapapillary epidermal plate. The dermoepidermal junction appears as round to oval rings of dark cells measuring about $10\text{to}12\mu \mathrm{m}$ surrounding the bright dermal capillary loops.

Fig. 3

Phase 1 of epidermal wound healing. (a) Close-up digital photograph of the epidermal wound immediately after the removal of the epidermis. Wound exudate dominates the appearance of the wound bed. (b) FLSM image focused on the wound edge. Wound exudation appears as a bright homogenous acellular area due to accumulating fluorescein (left upper border). (c) When focusing on the wound bed, the keratinocyte layer of the stratum basale and papillary structures are observed. The keratinocytes show an irregular shape after the mechanical removal of the upper epidermis. Scale bars: $50\mu \mathrm{m}$ .

In phase 2 of the wound healing process, clinical evaluation reveals that the wound bed is already covered by a thin layer of keratinocytes [Fig. 4a ]. Corresponding FLSM evaluation demonstrates the presence of sparse clusters of corneocytes developing near the wound edge [Fig. 4b] and around hair follicles [Fig. 4c]. Phase 2 is completed after the formation of the first layer of corneocytes on the surface of the wound.

Fig. 4

Phase 2 of epidermal wound healing. (a) The wound bed is covered with the first layer of keratinocytes. (b) The first corneocytes are found at the wound edge growing into the epithelial defect. (c) Keratinocytes around hair follicles (dark gray, black) start to grow into the wound bed. Scale bars: $50\mu \mathrm{m}$ .

The clinical photograph of a representative wound in early phase 3 shows an almost reestablished skin surface aside from the ridges that are still missing [Fig. 5a ]. Corresponding FLSM images reveal that the corneocytes in early phase 3 [Figs. 5b and 5c] do not appear as organized as seen in healthy skin [Fig 2a], where corneocytes form a tight and uniform honeycomb pattern. Following FLSM evaluation in late phase 3, most corneocytes start to resume their uniform polygonal shape but still show uneven, bizarre-shaped corneocytes usually found in dry, chapped skin [Figs. 5b and 5c]. Figure 5d reveals an evenly distributed honeycombed pattern of uniform, polygonal-shaped corneocytes at the level of the stratum granulosum. Measurement of the entire epidermis at this time point using FLSM reveals the thickness of the normal epidermis adjacent to the former wound site, marking the completion of phase 3.

Fig. 5

Phase 3 of epidermal wound healing. (a) Close-up digital photography shows a completed tissue repair except the missing furrows of the epidermal skin. The wound is still partly covered with a small amount of encrusted exudate. (b) Several layers of polygonal corneocytes can be found at the entire wound site still showing an irregular shape. (c) At the level of the stratum corneum, bizarre-shaped, detached corneocytes resembling the aspect of keratinocytes are found in dry skin. (d) At the level of the stratum granulosum, a complete restoration of the typical honeycombed pattern can be observed. The complete restoration of the epidermis can also be validated by in vivo FLSM thickness measurements showing a thickness of $\sim 20\mu \mathrm{m}$ from the stratum corneum to the bordering stratum granulosum, corresponding to values of normal skin. Scale bars: $50\mu \mathrm{m}$ .

3.3.

Effects of Topically Applied Wound Ointment on Epidermal Tissue Repair

Figure 6 depicts the average wound healing phase of topically treated and untreated wounds for all six study participants over a time course of $8\text{days}$ . Interestingly, after the removal of the epidermal roof of the suction blister (phase 1), the formation of the first corneocyte layer (phase 2) occurs already around day 5 in the wound ointment–treated wound sites when compared to the untreated wound sites, where the corneocyte layer is not found before day 7. At the end of the experiment at day 8, all wounds treated with the ointment have reached phase 3, corresponding to a completely reestablished epidermis with all cell layers and a thickness comparable to the normal skin adjacent to the site of wounding. The reestablishment of all epidermal cell layers was documented by FLSM imaging of each layer from the skin surface down to the basal cell layer and by obtaining thickness measurements of the epidermis using FLSM in comparison to normal skin.

Fig. 6

Comparison of wound healing kinetics between treated and untreated wounds. Wounds were classified into three phases according to FLSM findings, as outlined in the Sec. 2. The specific phase of wound healing was determined for all six study participants, on days 1, 3, 5, 7, and 8, and the means of the wound healing phase were calculated for each group for all investigated time points. To compare the means of untreated and treated wounds, two-tailed Student $t$ -tests for paired data were performed (significance level ${}^{*}p<0.05$ ). $x$ axis corresponds to wound healing phase (1 to 3); $y$ axis corresponds to days (investigated time points).