Preserved extracellular matrix in non‐enzymatically detached cultured keratinocyte sheets bolsters the healing effects on grafted burn wounds
Authors: Alharbi Suzan , Niimi Yosuke , Wiener Dominique , Hawkins Hal , Popov Vsevolod , Osada Atsuyoshi , Sakurai Hiroyuki , Enkhbaatar Perenlei 
Published: April 2020

Cultured autologous keratinocyte sheets have been proposed for treatments of burn wounds. However, this method has not been successfully translated to clinical practice because of certain limitations, such as inconsistency of engraftment, incidence of infection, spontaneous blistering, and scar contracture. Enzymatic detachment of cultured keratinocyte sheets (KS) from culture dishes was attributed to these negative outcomes. To overcome this problem, we are testing a novel technology that allows the detachment of cultured keratinocyte sheets without using any enzymatic treatment. We compared efficacy of keratinocyte sheets cultured on 2 different dishes: temperature responsive (TRD) and conventional. Cells cultured on TRD were detached by temperature reduction (T‐KS), whereas the cells cultured on conventional dish were detached by enzyme, Dispase (D‐KS). We studied efficacy of these sheets (T‐KS and D‐KS) on both in vitro and in vivo burn wound models and characterized downstream mechanisms underlying differences between the two sheets.

In that study, we were found that T‐KS were significantly thicker, preserved more ECM and cytoskeleton proteins, when compared to D‐KS. Therefore, the integrity of T‐KS, tested using mechanical cell dissociation assay, was still intact, while D‐KS was fragmented to multiple pieces. This was correlated with significantly higher number of disassociated cells in D‐KS compared to T‐KS. Also, the reduction in cytoskeleton proteins were associated with significant shrinkage of D‐KS compared to T‐KS. We created in vitro burn skin wound healing model and tested the efficacy of both types of cultured KSs and investigated their proliferation and cellular mechanisms. We found that T‐KS had significantly higher rate of proliferation compared to D‐KS. Also, the activation of MAPK, was lower in the sheet detached by dispase treatment compared to temperature reduction.

We wanted to compare wound healing efficacy of cultured T‐KSs and DKSs in 3rd degree ovine burn wounds grafted with cadaver skin. We designed the experiments in such way that closely mimics clinical situation. The burned skin was excised 24 hrs postinjury, followed by grafting with sheep cadaver skin. The formation of KSs was completed at the time of grafted cadaver skin rejection, thus enabling timely transplantation of KSs to the wounds. By using planimetric analysis, we found that wounds treated with T‐KSs had a significantly higher epithelization rate, firmer dermal‐epidermal junction when compared to D‐KSs and control wounds at 14 days of grafting. The vascularization rate tended to be higher in the T‐KSs‐treated wound beds compared to the D‐KS wounds. Next, we have measured a potent growth factor KGF, which is highly expressed in early wound healing. The level of KGF, in the wounds treated with T‐KS tended to be lower reflecting more mature wounds treated with T‐KSs.

Taken together, above results suggest overall superior quality of T‐KSs and immature sheet integrity and fragility in D‐KSs were linked to use of enzyme for detachment that disrupted ECM.