**3. Results**

## *3.1. Clinical Outcome*

The clinical picture of Patient One's massive pressure ulcer prior WMCS treatment is shown in Figure 1A (a–i). A clear change in wound edges with an obvious reduction in the wound's dimensions and total area was observed after a period of ~3-month treatment Figure 1A (c). A 50% of wound closure was achieved after a 6-month period of treatment Figure 1A (d–f), while a more than 90% wound closure and regrowth of normal skin was seen by the end of a total of 8 to 9 month WMCS treatment Figure 1A (g–i).

(**B**) 

**Figure 1.** (**A**) (a–i): Serial photography of the clinical outcome of Patient 1 after WMCS treatment. (**B**) Presentation of photographic wound closure percentage of Patient 1. Wound healing is expressed as percentage wound area closure relative to original size [(wound area)/(original wound area)].

Measurement of the wounded area is one of the key aspects in the assessment of the healing process, since it can indicate healing improvement. Figure 1B shows the percentage (%) of wound closure for Patient 1 from photographs taken immediately after treatment with WMCS. As shown, wound healing started gradually after WMCS treatment, with an obvious macroscopic improvement even at the end of the 1st month of treatment. At approximately six months of treatment, a 50% wound closure was achieved, while by the end of the 9-month treatment a greater than 95% of wound closure was observed as compared to the initial wound area before WMCS treatment.

For Patient 2, the clinical picture of the wound prior WMCS treatment is shown in Figure 2A (a). After three sessions, healthy new epithelial tissue was visible in the wound bed and at the wound edge, Figure 2A (b). A clear reduction in the wound's dimensions and total area was achieved after 6 sessions, Figure 2A (c), allowing for no dressing changes. Continued decrease in scar tissue and regrowth of normal skin were observed from day 9 to day 15, Figure 2A (d–f), with improved wound contraction and significant re-epithelization.

**Figure 2.** (**A**) (a–f): Serial photography of the clinical outcome of Patient 2 after WMCS treatment. (**B**) Presentation of the photographic wound closure percentage of Patient 2. Wound healing is expressed as percentage wound area closure relative to original size [(wound area)/(original wound area)].

Figure 2B is showing the percentage (%) of wound closure for Patient 2 as a factor of time treatment with the WMCS technology from photographs taken after treatment. As we can see, this wound started gradually to heal immediately after WMCS treatment, with more obvious changes in wound dimensions after a week (Day 6 to 7). An approximately 50% of wound area reduction in size was observed at day 8 to 9, whereas at day 15 with WMCS treatment, the wound was almost entirely healed.

## *3.2. Immunohistochemical Analysis*

Monitoring healing progress by tissue biopsies of the wounded area is the hallmark of the evaluation of an active and proper wound healing process. Thus, tissue biopsies were immunohistochemically analyzed in order to identify potential cellular events upon WMCS treatment that are indicative of a restart of wound healing. Figure 3A–C shows the Hematoxylin and Eosin stain of tissue biopsy from Patient 1. In detail, Figure 3A depicts signs of active granulocyte aggregation with some signs of inflammation after 1-month WMCS treatment sessions. A suppression of inflammation upon WMCS treatment (2-month) as evidenced followed by an increase in myofibroblastic activity and a reduced granulocyte aggregation, is demonstrated in Figure 3B. After 3-month of WMCS treatment (Figure 3C), microscopic examination revealed further established myofibroblastic activity and almost totally diminished granulocyte aggregation.

Increased numbers of mast cells (c-kit antigen stain, panels D–F) are present at early stages of wound healing, indicating an active process of inflammatory reactions (Figure 3D). WMCS treatment seems to a ffect the activation of mast cells, as an increased number of visible mast cells is observed after 1-month WMCS treatment (Figure 3D), ultimately leading to the observation of few scattered mast cells between 2-month and 3-month of WMCS therapeutic treatments (Figure 3E,F).

Tissue biopsies from Patient 2 were stained apart from H&E stain with the Masson's trichrome stain and the c-kit antigen for collagen formation, myofibroblastic proliferation and mast cell existence after WMCS treatment. As shown in Figure 4A–C, a gradually increased amount of collagen deposition during the healing process is observed after WMCS treatment.

**Figure 4.** (**A**–**C**) Masson's trichrome stain of tissue biopsies from Patient 2. Masson's stain indicating collagen formation and myofibroblastic proliferation after WMCS treatment.

Moreover, WMCS enhances myofibroblastic proliferation (Figure 5A), ye<sup>t</sup> with a relative focal increase of mast cells after three Days (Figure 5B). Finally, after six Days of WMCS treatment few scattered mast cells are seen as revealed by the c-kit antigen (Figure 5C).

**Figure 5.** (**A**–**C**) Immunohistochemical analysis of tissue biopsies from Patient 2, showing increased myofibroblastic activity, with a relative focal increase of mast cells (H&E stain 5A and 5B, respectively), and c-kit (CD117) antigen stain showing reduced CD117+ mast cells over WMCS treatment (5C).
