The overall goal of this method is to quantify the dynamic mechanical stiffness and build up of drying stresses in ex vivo samples of human stratum corneum, after different chemical treatments. This method can help answer key questions in the biomechanics and cosmetic science fields, such as how the composition of of the stratum corneum or treatment of the skin tissue with a personal care products can influence, it's mechanical properties and dynamic drying behavior. The main advantage of this technique is that it offers a more rapid alternative to other mechanical assessment methods, requires significantly less skin tissue and provides more physiologically relevant drying by preventing evaporation from the sample underside.
To prepare elastomer coated coverslips, first make Sylgard 184 curing agent with base solids at a base to curing agent ratio of 55 to one, until it is homogenous. Six grams in total are needed. Next, de-gas the solution.
Then use the solution to coat the coverslips on a spin coater. Using a wide bore pipette, drop one milliliter of the mixture centrally on a coverslip. Then spin coat the coverslip at 2, 000 rpm for 60 seconds.
Make five or six coated coverslips in this manner. To cure the coverslips, keep them in a 60 degree Celsius oven for 12 hours. The next day, measure the elastomer films thicknesess.
Use a razor blade to lift one edge of the elastomer film off the coverslip. Then use an indelible marker to mark the top side of the elastomer film and to mark the treated coverslip beneath. Now, using an inverted microscope, measure the Z height between the focal planes of the marks to measure the films thicknesses.
Follow standard procedures to prepare the SC tissue. To make samples, take six millimeter diameter punches from the SC.On the samples, place a mark at the center of the outmost face using an indelible marker. Next, deposit fluorescent marker beads onto the SC surface by floating the samples in 15 milliliters of water, containing about 90 microliters of bead concentrate.
Use gentle agitation for 30 minutes and adjust the bead concentration as needed. After the treatment, immerse each sample in water to mount it onto the elastomer. Position a coated coverslip at a shallow angle, 15 to 30 degrees under the SC with the elastomer towards the bottom side of the SC.Then lift the coverslip from the water with the SC attached above, thus smoothly laminating the SC sample without wrinkles or trapped air.
Next, dry the mounted samples at ambient conditions for about an hour. Once dried, transfer the samples to a hermetically sealed chamber containing a dish of water. Allow the samples to equilibrate to the humidity for 24 hours.
The next day, mount a sample to a perfusion chamber for a prolonged imaging session. Place the perfusion chamber over the substrate and seal the edges of the perfusion chamber to the elastomer using vacuum grease. Then record fluorescent and transmitted light images with a field of view large enough to capture the full SC sample.
Over the next 16 hours, take an image every 10 minutes. In a chemical fume hood, load a small plastic cap with one milliliter of silane. Transfer the cap into a sealed container with one elastomer lined coverslip, not in direct contact.
Allow them to incubate at room temperature for five hours. While waiting, prepare 20 milliliters of bead solution according to the text protocol and then pour it into a 10 centimeter dish. After five hours, slowly place the silanated substrates elastomer side down, into the bead solution.
Load two substrates into each dish and let the substrates react with the beads for 45 minutes. Later, using tweezers, remove the substrates and rinse them in distilled water to remove the unbound beads. Then dry them under a gentle stream of compressed air and store them in a sealed, opaque box.
Using the previously described technique, transfer the SC samples onto a treated substrate in a water bath. Immediately after mounting the SC, apply a five microliter drop of un-diluted fluorescent marker bead solution onto it. Then allow the SC to dry for 60 minutes.
Now, measure the SC thickness using the microscope with a 40x subjective lens in a perfusion chamber. Measure the thickness of SC over time, using a remote focus accessory to record the difference in Z height between the two bead layers. One layer is at the SC substrate interface and the other is on top of the SC.Using the described techniques, an SC sample was prepared and coated with fluorescent beads.
After 16 hours of drying, at 25%relative humidity, displacements were measured. Due to the circular symmetry of the samples, these displacements were azimuthally averaged. Throughout drying, azimuthal displacements remain small.
Radial displacement profiles however, increased monotonically from the center to the edge and grew in magnitude until an equilibrium was reached. Profiles recorded at 30 minute intervals show the time evolution of in-plane displacements. The average SC thickness decreased during drying, primarily over the first two hours.
The drying dynamic was then analyzed by fitting the displacement profiles to a linear, elastic contractility model using a minimum least squares approach. From the model, the contractual drying stress was extracted, as well as the elastic modulus. Both parameters increased rapidly over the first two hours of drying.
While attempting this procedure, it is important to remember to adhere the samples onto the substrate completely. Following this procedure, other methods like lipid staining can be performed in order to answer additional questions such as how particular cosmetic treatments alter tissue lipid composition.
