Demonstrate

Cell Line Burn Standardization Assay

In this in vitro burn model, an increase of cell injuries and changes in cell viability and morphology is observed in relation to the time exposed to hot HBSS, compared to the controls and determined with MTT assay and microscope visualization. The damage done by the hot HBSS causes apoptosis and detachment of cells in the culture flask. This analysis allows us to proceed with the evaluation of TecTissue performing the same assay and applying the designed treatment. The importance of this standardization lies in the fact that it establishes how to perform future assays involving burns injuries, this information will be useful for future lines of investigation regarding burn assays.

UNIVERSUM 1 — Figure 1. Cell Visualization of different times of burn treatment using 60ºC HBSS in L-929 cell line

Burn assay with treatment

In this in vitro model is possible to observe several factors first, that co-cultures effectively have enhanced cell proliferation and metabolism in comparison with the controls in the different treatments.
In the treatment involving both cell lines, we can notice that human mesenchymal cell lines differentiate to fibroblasts, due that this is the normal human process in tissue regeneration and also due to the liberation of TGF-ß from stem cells [1].
The behavior seen between nano-encapsulated leptin and non encapsulated is that we can see a better proliferation in the first one, due is slowly released into the matrix allowing cells to absorb it and enhance proliferation, without inhibiting the metabolic path due to an excess of it [2]. In this assay it is seen that leptin is causing an acceleration of cell metabolism because of a high intake of glucose which is converted in citric acid, acidifying the medium, this is seen by the change of color in it [3]. Therefore, our thesis that TecTissue will enhance regeneration. Even though we demonstrated the efficiency of TecTissue, it is important to mention that within 72 hours, the confluence is the same on treated wells and untreated wells, which means that the effects of our treatment in the cell lines are in short term. Further analysis should be made to determine whether it is possible to increase the efficiency of the treatment considering the areas of opportunity identified after the assay.

Some aspects to consider are that even though the leptin, heparan, and collagen were studied, none of them were evaluated separately, which means that further analysis should be made to determine the activity of each element. As stated in the corresponding sections, each molecule plays a specific role in our project and the combination of them represent a model treatment for burn injuries. An example is that chitosan is seen that alone could induce fibroblasts regeneration and proliferation [2].

Cell Culture in BOB

The results demonstrate that porous-collagen-based scaffolds and PDMS membrane are effective for cell culture and tissue engineering. The images obtained from the microscope allow us to conclude that employing these elements can sustain a co-culture in our hardware. This is supported by the immunofluorescence assay that allows us to see the correct attachment of the cells. Finally, our automated system proves that it is capable of maintaining a sterile cell culture

TecTissue

Our results demonstrate that our novel treatment will enhance tissue healing and cell proliferation, incorporating heparin, collagen, and tenascin as scaffold and leptin as a growth factor. All this nano-encapsulated in Chitosan to ensure good cell incorporation. This treatment will save people lives and reduce burn mortality

TecTissue: heal a wound, heal the world.

References