Dimensiones de los movimientos.

As the lipid disordering in SC of the SkinBaR model was higher compared to the conformational ordering in SC in the clinical setting, the relation between ceramide composition and lipid conformational ordering in (regenerated) SC was examined. This will provide insight in how the CER composition contributes to the lipid ordering. To obtain insight in the contribution of parameters related to ceramide composition, these were used to make a predictive model for the lipid ordering (i.e. CH2 stretching peak

position). We included the following ceramide related parameters in a LMM (Table S8): C34 and EO percentage (together being an MCL predictor) and the following subclass molar ratios: (dS+P+H)/S (excluding EO ceramides) and N/A, the former ratio correlated with barrier function in a previous study.18 _{For more details about the subclass ratios,}

see Supplementary Methods. Figure 4B depicts the actual CH2-peak position compared

to the predicted position. The r2 _{indicates that 83% of the variation was explained, with}

normally distributed residuals. This model showed that the combination of these CER- related parameters could, to a large extend, predict the lipid conformational ordering in SkinBaR and in vivo samples.

Discussion

In general, skin models have been used to i) model either healthy or diseased skin,7,19,20

ii) examine treatment by formulations or active compounds,8,9 _{iii) to study the dynamic}

process of SC regeneration,11,12 _{iv) to study biological processes in the skin, and v)}

for screening of toxicity and irritation of compounds. Previously, we developed the SkinBaR model that has the potential to be used for multiple of these purposes.13

However, the regenerated SC CER composition and lipid organization of this model have not been evaluated in detail and the correlation to an in vivo tape-stripping method is unknown. Therefore, the aim of this study was to examine to what extent the lipid matrix properties of the ex vivo and in vivo compromised skin models are comparable, and to determine if ex vivo models can (partly) replace clinical studies with regards to skin barrier repair.

In general, the changes in CER levels and lipid properties in the RegSkinBaR _{samples were}

very similar, but more pronounced than in the RegIn-vivo _{samples as discussed below.}

i) Both regenerated SC models showed a decrease in CER P subclasses and increase in S subclasses compared to control SC samples. This might be caused by an imbalance in activity of the enzymes acid sphingomyelinase and

β-glucocerebrosidase, both involved in post-synthetic modification of CERs.21

Although the expression of these enzymes was not affected in the SkinBaR model,13 _{the activity can be altered.}22 _{With regard to properties of the CER}

chains, both the RegSkinBaR _{and Reg}In-vivo _{samples showed a decreased CER MCL,}

and an increase in levels of C34 CERs, while the level of unsaturated CERs was predominantly increased in RegSkinBaR _{SC. In agreement with the elevated level}

of unsaturated CERs, the expression of Stearoyl-CoA desaturase was increased in the SkinBaR model.13

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model was the change in level of EO CERs, which was increased in the RegIn- vivo _{samples and decreased in the Reg}SkinBaR _samples.

iii) The lipid ordering was not significantly affected by regeneration in both in vivo

and ex vivo conditions. This might be due to less deep stripping in the present study compared to previous studies. Previously, lipids in the regenerated SC of the SkinBaR model were less ordered when almost all SC was removed.14

Although the difference in CH2 stretching peak position between CtrlIn-vivo and

CtrlSkinBaR _{was significant, different spectrum collection methods could have}

played a role. However, as the lipid composition could be used to accurately predict the lipid ordering, the difference mainly originated from a difference in lipid composition. These variances in lipid composition might have originated from the different method of SC sample collection, i.e. only tape 5-8 for in vivo conditions vs whole SC for ex vivo conditions, a similar difference as performed in collection of the FTIR data.

The tape-stripping method has been developed and used for several purposes, such as to specifically study the dynamic process of SC production,23 _{to determine the}

penetration of topically applied products,24 _{and to increase the bioavailability of these}

products in the deeper epidermal cell layers. As barrier function recovery of stripped healthy skin takes at least 14 days,25 _{tape-stripped skin is also a suitable model to study}

skin barrier repair. Previously, it has been shown that in both in vivo and SkinBaR skin the proliferation rate immediately after barrier disruption is higher than after a recovery period of several days.7,13 _{However, in the SkinBaR model, the proliferation rate was}

at its normal level after 8 days of culturing,13 _{whereas in in vivo skin, the proliferation}

rate remained elevated for at least 10 days.7

It has been shown that tape-stripping healthy skin induced parakeratosis,7 _{and induced}

an inflammatory response by secretion of cytokines.26,27 _{Furthermore, in both AD and}

psoriasis pro-inflammatory cytokines are secreted.28,29 _{Therefore, tape-stripped healthy}

skin has been used as an alternative for several inflammatory skin diseases.7 _During

skin barrier repair of healthy in vivo skin, this inflammatory response slows down the repair process.30 _{The lack of this systemic response in the SkinBaR model therefore}

substantiates the rapid barrier repair process. This also indicates that, during skin barrier repair, the in vivo skin is closer to skin homeostasis.

In contrast to the differences in proliferation rate and cytokine excretion, the expression pattern of proliferation proteins is very similar during in vivo and ex vivo skin barrier repair.7,13

When comparing the lipid properties of regenerated SC to that in SC of inflammatory diseased skin, many similarities can be observed. When compared to AD skin, these are i) similar changes in CER subclasses,15 _{ii) a decreased MCL,}31 _{iii) an increase in levels of}

C34 CERs,15,31 _{and iv) an increased level of unsaturated lipids.}32 _{Most of these changes}

correlated with an impaired skin barrier function.3,15,17 _{Although the degree of changes}

vary, many of these alterations in CER composition have also been observed in other inflammatory skin diseases like psoriasis, and Netherton syndrome.33,34 _{Additionally, a}

less dense lipid packing observed in RegSkinBaR _SC14 _{also corresponds to findings in SC}

of these inflammatory skin diseases: a higher fraction of lipids adopting a less dense hexagonal lateral packing has been reported.35

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samples, and multiple samples of the same donor were used to generate the cultured and regenerated samples. Ideally, skin of the same participants is used for both the

in vivo and SkinBaR experiments. However, the SkinBaR samples are difficult to culture with small biopsies. Furthermore, multiple biopsies of one donor would be required. This is difficult to achieve. Lastly, the lipid properties of both models were compared to skin of inflammatory skin diseases based on data found in literature. In future experiments, the SkinBaR model should be directly compared to non-lesional and lesional diseased skin in the same experiment to minimize difference between measurements.

In conclusion, this study shows that the changes in lipid properties in both the RegSkinBaR

and RegIn-vivo _{models are very similar and mimic the lipid properties in inflammatory}

skin diseases. This concerns changes in CER subclass profiles, MCL, level of CER unsaturation, and conformational ordering. Therefore, the SkinBaR model can be used to predict the in vivo response with regards to the lipid composition after application of topical barrier repair treatments aiming to restore the normal CER composition. By doing so, the need for clinical studies is reduced.

Materials and methods