## 3 Results and Discussions
### Cell Culture and Treatment
#### 3.4.1 Cell Viability and Proliferation Assay (MTS) of Samples
Wound healing is a dynamic process where activation, proliferation and migration of fibroblasts are the primary steps involved, along with other microenvironmental factors [77; 78]. In the current study, normal human dermal fibroblasts (NHDFs) were treated with both different concentrations of mulberry extract (25, 50, 100 and 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) and mulberry extract included in liposome (25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for evaluation of cell migration for 0, 12, 24, 36 and 48 h (Figure 4). The images were captured using the CytoSMART Lux3 BR(r) device. The quantitative parameters such as wound closure by area (Wnd_A) (%) were calculated using CytoSMART Lux3 BR(r) and ImageJ software and the results are presented in Table 5.  
Figure 4: The evolution over time of the migration of normal dermal fibroblast (NHDF) for healing the wound formed in vitro by the scratch method depending on the applied treatment (different concentrations of mulberry extract (25, 50, 100 and 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), or liposomal formula (L and MnL) along with CTRL and positive control (Ala_50). NHDF dermal cells are represented in white and blue color shows the in vitro simulated wound that was not healed. The images obtained with the CytoSMART Lux3 BR\({}^{\circledR}\) device (Figure 4) indicate the evolution over time of the closure of the \"scratch\" caused in vitro depending on the applied treatment. The results showed that the most effective were the treatments with multiberry extract in a concentration of 25 ug/mL (MnE_25) and mulberry extract included in liposomes (MnL), the in vitro coverage area of the scratch was 93.81% and 99.72% respectively in 48 h.  
According to the data in Table 5, it can be seen that in the case of liposomes loaded with mulberry, a statistically significant (\(p<0.05\)) increase in the wound closure% is obtained starting from the time of 24 h compared to sample L. The wound healing process is an extremely complex one, involving a series of factors that act in four cascade-type stages [79; 80]. The first stage is the vasoconstriction stage, namely the stage of stopping the bleeding and self-cleaning of the wound. In this stage, at the level of the wound, the transformation of prothrombin into thrombin, of fibrinogen into fibrin and subsequent formation of a crust made of platelets takes place. The next stage is the pro-inflammatory one, which is generated by the immune system, the main characteristic being vasodilatation with an increase in the concentration of oxygen and pro-healing elements at the level of the wound. Also in this stage, phagocytic cells are concentrated at the level of the wound, which practically clean the wound and act as antimicrobial elements. The MnE extract is a rich source in phenolic acids, flavonoids, phytocompounds which are proven to have antimicrobial activity and can be considered a stimulator of wound scar formation [10; 11; 12]. The proliferation phase is the third stage of wound healing. This stage begins with the phenomenon of migration of dermal fibroblasts followed by the formation of the cell matrix and re-epithelialization [81]. The process of cellular migration leads in the first phase to the covering of the wound in a monolayer of dermal cells which then end up proliferating and overlapping in several layers. An extract's composition can influence neoangiogenesis and growth factor stimulation [73; 74; 75; 76]. Thus, we consider that the MnE extract contributed through the phyto-chemical composition and at this stage a different stimulation of the healing process can be observed depending on the form of extract applied (MnEs and MnL) but also depending on the concentration applied (MnE extract 25 ug/mL having a significantly different effect compared to the rest of the applied concentrations).