The application of amniotic membrane (AM) on chronic wounds has proven very effective at resetting wound healing, particularly in re-epithelialization. Historically, several aspects of AM effect on wound healing have been evaluated using cell models. In keratinocytes, the presence of AM induces the activation of mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) pathways, together with the high expression of c-Jun, an important transcription factor for the progression of the re-epithelialization tongue. In general, the levels of transforming growth factor (TGF)-β present in a wound are critical for the process of wound healing; they are elevated during the inflammation phase and remain high in some chronic wounds. Interestingly, the presence of AM, through epidermal growth factor (EGF) signaling, produces a fine-tuning of the TGF-β signaling pathway that re-conducts the stalled process of wound healing. However, the complete suppression of TGF-β signaling has proven negative for the AM stimulation of migration, suggesting that a minimal amount of TGF-β signaling is required for proper wound healing. Regarding migration machinery, AM contributes to the dynamics of focal adhesions, producing a high turnover and thus speeding up remodeling. This is clear because proteins, such as Paxillin, are activated upon treatment with AM. On top of this, AM also produces changes in the expression of Paxillin. Although we have made great progress in understanding the effects of AM on chronic wound healing, a long way is still ahead of us to fully comprehend its effects.
Unsuccessful wound closure in chronic wounds can be linked to altered keratinocyte activation and their inability to re-epithelize. Suggested mechanisms driving this impairment involve unbalanced cytokine signaling. However, the molecular events leading to these aberrant responses are poorly understood. Among cytokines affecting keratinocyte responses, Transforming Growth Factor-β (TFG-β) is thought to have a great impact. In this study, we have used a previously characterized skin epidermal in vitro model, HaCaT cells continuously exposed to TGF-β1, to study the wound recovery capabilities of chronified/senescent keratinocytes. In this setting, chronified keratinocytes show decreased migration and reduced activation in response to injury. Amniotic membrane (AM) has been used successfully to manage unresponsive complicated wounds. In our in vitro setting, AM treatment of chronified keratinocytes re-enabled migration in the early stages of wound healing, also promoting proliferation at later stages. Interestingly, when checking the gene expression of markers known to be altered in TGF-β chronified cells and involved in cell cycle regulation, early migratory responses, senescence, and chronic inflammation, we discovered that AM treatment seemed to reset back to keratinocyte status. The analysis of the evolution of both the levels of keratinocyte activation marker cytokeratin 17 and the spatial-temporal expression pattern of the proliferation marker Ki-67 in human in vivo biopsy samples suggests that responses to AM recorded in TGF-β chronified HaCaT cells would be homologous to those of resident keratinocytes in chronic wounds. All these results provide further evidence that sustained TGF-β might play a key role in wound chronification and postulate the validity of our TGF-β chronified HaCaT in vitro model for the study of chronic wound physiology.
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