ANTI-ACNE
have begun to emerge showing a good relevance and pertinency. The challenge with trying to grow skin commensals or potentially other probiotics on such in vitro tissue models is that they can overwhelm the tissue growth and thus simply become a contaminant in the model. However, recently Gruber et al reported
on the topical treatment of 3D RHE models with a probiotic treatment containing viable Lactobacillus plantarum cultures that demonstrated an ability to upregulate elastin in these tissue models.14 In addition, Meloni et al have described an in
vitro testing model in which the skin commensal microbes, C. acnes and Malassesia restricta are grown on RHE. The model assayed the influence of skin commensals to influence skin barrier structure, cellular proliferation and differentiation and expression of anti-microbial defences.15 In the present work, the model described
by Meloni et al was modified to examine the influence of C. acnes to modulate skin inflammatory responses including effects on TNF-a and Caspase-1. By demonstrating that the topical
application of C. acnes could upregulate these two critical skin inflammatory markers associated with inflammatory acne lesions, it was possible to then investigate the influence of a monographed acne treatment, salicylic acid, and a newly developed pre-solubilized form of azelaic acid alone and in combination with each other. In addition, samples of dissolved azelaic acid
derived from the pre-solubilized technology were compared against suspended azelaic acid as is typically found in commercial gels.
Materials and methods Reconstructed human epidermis The 3D human epidermis full thickness EpiDermFT (EFT-400, MatTek Company; production site: Bratislava, Slovakia) consisting of a fully differentiated epidermis with cornified epidermal layers: keratin 5 expressed in basal cells, involucrin and keratin 10 expressed in the spinous and granular layers.
Epidermal Arrival Day 1 Negative Control
Untreated Control Active Treated
Figure 1: Assay protocol schematic
www.personalcaremagazine.com April 2024 PERSONAL CARE 48h 48h 72h
TABLE 1: SUMMARY OF INGREDIENTS TESTED Active Ingredient
5% Solubilized Azelaic Acid 5% Suspended Azelaic Acid 10% Solubilized Azelaic Acid 10% Suspended Azelaic Acid 1% Salicylic Acid 2% Salicylic Acid 1% Salicylic Acid/5% Azelaic Acid 2% Salicylic Acid/5% Azelaic Acid 2% Salicylic Acid/10% Azelaic Acid
The dermal compartment is composed of
a collagen matrix containing viable normal human dermal fibroblasts (NHDF). The epidermal and dermal layers are mitotically and metabolically active and exhibit in vivo- like morphological and growth characteristics which are uniform and highly reproducible. A well-developed basement membrane is present at the dermal/epidermal junction. The tissues have been cultured on
specially prepared cell culture inserts (surface 1 cm2
) where all biological components of
the epidermis and the culture medium are tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. The tissues were shipped at 4°C in 24-well plates containing a nutrient agarose gel and may be stored at 2-8°C for up to six days prior to use. Cultures can be continued for up to two
weeks with good retention of normal epidermal morphology. Immediately after arrival of the test system in the laboratory, data sheets enclosed with the batch, shipment integrity, and color and temperature of the agar medium used for transport were checked. The tissues were then removed from the agarose nutrient solution under a sterile air flow cabin. The inserts were rapidly transferred to
12-well plates previously filled with culture medium without antibiotics and incubated at 37°C, 5% CO2
, saturated humidity. The day of Untreated Tissues
107
the test, the medium was replaced with fresh growth medium without antibiotics.
Test materials All test materials were provided by Vantage and are summarized in Table 1. Curazelic® 44 [INCI: Cocamidopropyl Dimethylamine (and) Azelaic Acid], is a newly developed, pre- solubilized form of azelaic acid that contains 44% azelaic acid dissolved in a tertiary amine emollient, and it was further diluted in DI water to make 5% and 10% solubilized azelaic acid for the test.
The salicylic acid employed was also
provided as a pre-solubilized solution in the same emollient (Curcylic 40). Similarly, Curcylic 40 was further diluted in deionised to make 1% and 2% salicylic acid for use. 10% suspended azelaic acid was a commercially available product (Azelaic Acid Suspension 10%, The Ordinary, USA).
Growth of bacterial suspension and tissue treatments Before the assay, the test strain C. acnes ATCC 11827, was sown on agar culture medium, starting from glycerinate culture stored at <-70°C and incubated at 37°C, for one to two days in anaerobic conditions to check its normal colony morphology. A second subculture was performed to use fresh culture.
Preparation of microbial suspension and colonization procedure The day of the test, the bacterial suspension was resuspended in sterile saline solution, adjusting the concentration level of 107-108 CFU/mL by OD reading (via spectrophotometer), considering an application volume of 60μL. The bacterial suspension was then used for
the FT skin colonization procedure: 6μL of lipid mix (olive oil and neutral TAG mixed 1:1) on the tissues surface added with 60μL of C. acnes ATCC 11827, 107-108 CFU/mL for 72h at 37°C, 5% CO2
and 90% RH. A viable count on agar plates
was performed to check the starting inoculum concentration.
Colonization with
C.acnes ATCC 11827 Cultural media collection for ELISA assays Test Item Treatments Maintenance media w/out absorbance change
24h 24h
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154
