Repair attributes are key components of an effective skin care formula. The study described in this article focuses on the determination of the in vitro capability of two cosmetic raw materials to promote cell repairing induced in an in vitro experimental model represented by human skin keratinocytes in monolayer. The repairing efficacy was assessed by the product’s ability to promote the healing of damaged tissue in the in vitro system. The test was carried out by using cell cultures in the G0 phase of cells cycle in which the sample was added at different concentrations (1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%) to study the proliferation activity.

Biollagen SSE collagen is derived from Pichia yeast using a patented, high-density fermentation and highly efficient separation and purification process. The samples were tested at the concentrations of 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% before starting the proliferation test as a cytotoxicity assay and no cytotoxicity was detected at the end of the treatment.

Procedure:
The proliferation test was carried out by using:

• Human skin keratinocytes (ATCC PCS-200-010);
• Test item concentration: 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% in culture medium (DMEM); and
• Cell cultures without any treatment as negative control reference (CTR -) to calculate cell proliferation.

The cells were plated in a plate of 96 with incomplete culture medium (missing bovine fetal serum). This induces the phase of cellular quiescence. After 24 hours, the concentrations of the product to be tested were prepared in complete medium, in order to restart the cells from the G0 phase of the cell cycle. The negative control (CTR-) is set up with only complete medium.

Culture medium containing tested product at concentrations was added to the wells containing cells in the G0 phase of the cell cycle. Cells were exposed to the product for 24 and 48 hours. At the end of incubation period, MTT coloration was performed in order to evaluate cell viability and proliferation index compared to untreated control (CTR, 100% viability for definition).

MTT-medium is prepared by adding 15mg of MTT to 30ml of culture medium. After exposure of cells to the test items, they were washed with 200μl of PBS. After removal of the washing solution, 200μl of MTT-medium were added to each culture well and then incubated for 3 hours at 37°C and 5% CO2. At the end of the incubation period, the MTT-medium was removed and 200μl of isopropanol were added.
The plate was shaken on a rotatory plate for 30 minutes, in order to ensure that all the crystals have dissolved from the cells and have formed a homogeneous solution. The absorbance was measured at 540nm on a microplate reader. The results are expressed as % cell viability compared to an untreated control cell culture.

Method
Statistical analysis by T-test was conducted between the samples and the untreated negative control as well as between the two samples, Biollagen SSE and marine collagen. The variations are considered significant for p<0.05.

The repairing effect test was carried out by using cell cultures in which the sample was added after the wound creation to study the repairing activity and cell cultures without any treatment (untreated control). The samples were tested at the concentrations of 0.1%, after the cytotoxicity assay.

Cell cultures of human skin keratinocytes were used. The cells were put in 12 well-plates filled with Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with fetal bovine serum 10%. Cells were incubated at 37°C and 5% CO2 until full confluence. An artificial wound was mechanically created in the cell monolayer by sliding the tip of a pipette on the cell monolayer. For each experimental condition three replicas were performed.

The repairing effect of the test item was determined by acquisition of photographic images of the cell cultures at T0, T2H, T8H and T24H after the wound creation. At each experimental time, cells were visually checked using (Optika, XDS-2) at 4X and pictures with camera (TiEsseLab PrimoCam HD 5.0 with sensor Micron MT9P001) were collected. Pictures were analyzed with Image J software.

The distances between the wound margins for each picture were measured in μm and were recorded in three points of the wound length. The variations on wound margins were analyzed by T-test. Some representative images of the wound healing progress are reported for the different experimental conditions at all the monitored times.

Results
Both products resulted effective in increase cell proliferation on the considered in vitro system, however, under the same period and concentration conditions, Biollagen SSE shows a more prominent value-promoting effect than marine collagen. In 24h, the cell-promoting rate at the lowest concentration of Biollagen SSE is higher than marine collagen by 14.44% and the cell proliferation rate at the highest concentration of Biollagen SSE is higher than the marine collagen by 17.08%. Moreover, in cell proliferation, the 0.005% concentration of Biollagen SSE has similar effect as 1% concentration of marine collagen. Therefore, Biollagen SSE in promoting cell proliferation is more significant than marine collagen.

According to the obtained results, it is possible to highlight that the treatment of cell cultures with Biollagen SSE and marine collagen at 0.1% significantly promotes wound repair by making it faster. During the same period of time, Biollagen SSE shows more promise in cell repairing than marine collagen. The 24 hour experimental results show that Biollagen SSE’s cell repair percentage is 21.1% higher than marine collagen, and it is obtained by T test. In conclusion, Biollagen SSE has a faster onset of action and there is a significant difference within two hours. Therefore, Biollagen SSE is more effective than marine collagn in cell repairing. In particular, Biollagen SSE shows better repairing performances than marine collagen at all experimental times (p value < 0.05 at T0, T2, T8, T24) in the considered experimental system.

Conclusion
According to the data obtained and reported in this article, in the considered in vitro system, we can assess that the treatment of cell cultures with Biollagen SSE and marine collagen enhances cell proliferation and the treatment of considered cell cultures with Biollagen SSE and marine collagen enhance the healing of the wound.

According to tested experimental patterns, Biollagen SSE significantly enhances proliferation capability and “skin repairing” of cell culture.

In the specific in vitro model, Biollagen SSE shows a more prominent cell-promoting effect than marine collagen. The 0.005% Biollagen SSE has the same cell proliferation rate as 1% marine collagen.

In addition, in terms of cell repairing, compared with marine collagen and the control group, Biollagen SSE has a faster onset and has obvious effects within two hours. In summary, Biollagen SSE has a clear advantage over marine collagen in promoting cell proliferation and cell repairing. 

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