72 SKIN MICROBIOME
pro-inflammatory cytokines and chemokines when exposed to irritants that cause sensitisation to the skin. In in vivo human skin, these biomarkers are able to trigger immune cascades that active immune cells such as macrophages and T-cells to respond to the source of irritation.12
Results Microbiology results Figure 5 shows the representative colony forming units (CFU) per cm2
PRE-INOCULATION 48hr POST-INOCULATION POST-TREATMENT 24hr POST-TREATMENT
recovered from the Labskin-S post incubation. The left hand panel shows the CFU/cm2
of Cutibacteria spp. for the
Cutibacteria spp. in the microbiome sample used to inoculate the Labskin-S, the negative control (dPBS), the over-the-counter positive control and the test item (volunteers product of choice). The data shows that there is an increase between the initial inoculum
in the CFU/cm2
and the negative control, indicating that the Cutibacteria spp. is able to proliferate on the Labskin-S and that the dPBS does not have an anti-acne effect. When compared to the negative control,
both the positive control and test item contributed to a significant reduction in Cutibacteria spp. CFU counts. When compared to the positive control, the
test item contributed to a stronger reduction in Cutibacteria spp. CFU counts. This is shown in the right-hand panel additionally, where a log10 reduction in the CFU/cm2
is compared for the positive control and test item.
Immunology results Figure 6 shows the concentration of IL-1α pro-inflammatory cytokine detected in the experimental culture media (undernatent) analysed by ELISA. The lower limit of detection for the ELISA assay is shown by the black dotted line (3.9 pg/mL), and the upper limit of detection is shown by the red dotted line (250 pg/mL). The analysis software used us able to extrapolate data below and above these values. The uncolonized, untreated Labskin-S
control showed a very low level of IL-1α release, below the limit of detection. When compared to the uncolonized,
Cutibacteria spp. *
106 105 104 103 102 101 100
*
Figure 4: Macroscopic images of the skin constructs throughout the experimental procedure. The red arrows show the start of the formation of comedos (pimples) of the Labskin-S due to the concentrated presence of
C.acnes
untreated control, all test groups triggered a release of IL-1α pro-inflammatory cytokine. The negative control (dPBS) did show a high
level of IL-1α release. This could be due to the keratinocyte cells responding to the dPBS but is likely to be due to the physical process of applying the concentration of microbes in the microbiome. The over-the-counter positive control
showed a lower detectable level of IL-1α release compared to the negative control and test item. The test item contributed to the largest release of IL-1α pro-inflammatory cytokine.
0 -1 -2 -3 Inoculum Initial
Negative control
Postive control
Test Item
Figure 5: Left panel: Enumeration of Cutibacteria spp. (CFU cm-2) recovered from Labskin-S. Black dotted line represents the lower limit of detection (59 CFU cm-2). Right panel: Log10 CFU cm-2 difference between the test item and positive control. Black dotted line represents the threshold of significant log difference
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Uncolonised control
Negative control
Postive control
Test Item
Figure 6: Concentration of IL-1α release. Black and red dotted lines represent the lower (3.9 pg mL-1) and upper (250 pg mL-1) limits of detection of the assay, respectively
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350 300 250 200 150 100 50
Histological evaluation Full-thickness biopsy samples were taken from each Labskin-S construct and formalin fixed paraffin wax embedded. Sections were taken using a microtome and stained with haematoxylin and eosin (H&E). The distinct layers of the epidermis and
dermis are clearly visible, along with the differentiated layers of the epidermis, including a fully-formed stratum corneum as shown in Figure 7. As previously shown in the macroscopic
images, it is possible to create comedos on the Labskin-S constructs. A high concentration of
IL-1α
CFU cm2
Log10
Reduction TEST ITEM POSITIVE CONTROL NEGATIVE CONTROL
pg mL-1
Positive control Test Item
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