## 4 Materials and Methods  
### Study Population and Application Technique  
The study was conducted with 18 women (aged 42 to 62 years (51.18 \(\pm\) 5.18)), patients of the Department of Aesthetic Dermatology, for dermatological and cosmetic consultation. Included in the study were women whose skin on physical examination showed changes of photoaging, including hyperpigmentation, dilated sebaceous gland outlets, telangiectasias, gray and earth skin tone, flabbiness, dryness and roughness, loss of firmness, and the presence of deep wrinkles. Skin phototypes II and III (specific to a geographic region in Poland) and the desire to improve skin condition were also important factors. Exclusion criteria/interfering factors included a tendency to scarring and keloids, which preclude biopsy and micro-needling, as well as the presence of a vascular complex-ion, rosacea, and taking blood-thinning medications (a contraindication for mesotherapy with micro-needling). Other exclusion criteria included pregnancy, lactation, use of anti-aging cosmetic treatments 6 months prior to and during the study, as well as coexisting systemic diseases (hypertension, circulatory insufficiency, diabetes, epilepsy, viral infections such as herpes and bacterial infections of the skin, allergic conditions on the skin, sensory disorders, cancer up to 5 years after treatment). Contraindications to needleless mesotherapy also include the presence of metal implants, among other prostheses, and an implanted pacemaker. Interfering factors included UV/solarium exposure during the study from the time of the first biopsy, including the series of treatments and the biopsy taken 3 months after their completion. In addition, it was not advisable to use other alternative cosmetic treatments during the study and to introduce other/new cosmetic preparations with anti-aging activity into daily skincare than those recommended (containing snail mucus for groups A and C). The study was conducted during the autumn-winter period to exclude the effects of UVB radiation on the skin. Patients underwent a series of 6 mesotherapy treatments (at intervals of 14 days) of the facial skin and were randomly assigned into one of the groups--A, B, or C (n = 6--number of women in each group according to power test)--based on each method presented in Table 4. In addition, the patients completed a questionnaire. The study was approved by the Bioethics Committee of the Pomeranian Medical University in Szczecin (Resolution No. KB-0012/124/19). \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Group A (n = 6)** & **Group B (n = 6)** & **Group C (n = 6)** \\ \hline Micro-needle mesotherapy + the contents of an ampoule containing snail mucus 98.2\% (Nature Cosmetes, Healthy Skin line, Siewierz, Poland) & Micro-needle mesotherapy + the contents of an ampoule with 0.9\% NaCl solution (Polpharma, Starogard Gdärski, Poland) & Needle-free mesotherapy + the contents of an ampoule containing snail mucus 98.2\% (Nature Cosmetes, Healthy Skin line, Siewierz, Poland) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Scheme of application methods. In order to evenly treat the skin area with the head, the face was divided into treatment zones (Zone 1--forehead, Zones 2 and 3--cheeks, Zone 4--chin). The depth of needing for each area was regulated in the head. The following micro-needling depths were adopted for the respective treatment zones: 0.5-0.75 mm for Zone 1; 0.5-1 mm for Zones 2 and 3; 1-1.5 mm for Zone 4. The area around the eyes and mouth was needed to a depth of 0.25-0.5 mm. The specific ranges of treatment parameters were adjusted to the skin type and individual sensitivity. The skin was needed using a dermapen with a stamping technique. Then, to intensify the treatment of the selected area, the head was moved over the skin in vertical bands continuously without lifting it. Small, droplet-like bleeding was massaged into the skin. The above steps were repeated in each of the remaining treatment zones, applying the same volume of preparation to each zone.